608885.Full.Pdf

bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 Genomics, Molecular and Evolutionary Perspective of NAC 2 Transcription Factors

4 Tapan Kumar Mohanta1*ꞩ, Dhananjay Yadav2ꞩ, Adil Khan1, Abeer Hashem3,4, Baby Tabassum5, 5 Abdul Latif Khan1, Eslayed Fathi Abda_Allah6, Ahmed Al-Harrasi1*

6 1Natural and Medicinal Plant Sciences Research Center, University of Nizwa, Nizwa, 616, Oman 7 2Dept. of Medical Biotechnology, Yeungnam University, Gyeongsan, 38541, Republic of Korea. 8 3Botany and Microbiology Department, College of Science, King Saud University, P.O. Box. 9 2460, Riyadh 11451, Saudi Arabia. 10 4Mycology and Plant Disease Survey Department, Plant Pathology Research Institute, ARC, Giza 11 12511, Egypt. 12 5Toxicology laboratory, Department of Zoology, Raza P.G. College, Rampur, Uttar Pradesh, 13 244091, India 14 6Plant Production Department, College of Food and Agricultural Sciences, King Saud University, 15 P.O. Box. 2460 Riyadh 11451, Saudi Arabia. 16

17 ꞩ Contributed equally

18 *Corresponding author

19 Tapan Kumar Mohanta, E-mail: [email protected], [email protected]

20 Ahmed Al-Harrasi, E-mail: [email protected]

21 Abstract

22 NAC (NAM, ATAF1,2, and CUC2) transcription factors are one of the largest transcription factor 23 families found in plants and are involved in diverse developmental and signalling events. Despite 24 the availability of comprehensive genomic information from diverse plant species, the basic 25 genomic, biochemical, and evolutionary details of NAC TFs have not been established. Therefore, 26 NAC TFs family proteins from 160 plant species were analyzed in the current study. The analysis, 27 among other things, identified the first algal NAC TF in the Charophyte, Klebsormidium 28 flaccidum. Furthermore, our analysis revealed that NAC TFs are membrane bound and contain 29 monopartite, bipartite, and multipartite nuclear localization signals. NAC TFs were also found to 30 encode a novel chimeric protein domain and are part of a complex interactome network. 31 Synonymous codon usage is absent in NAC TFs and it appears that they have evolved from 32 orthologous ancestors and undergone significant duplication events to give rise to paralogous 33 NAC TFs.

34 Keywords: NAC transcription factor, Transmembrane domain, Chimeric NAC transcription 35 factor, Nuclear localization signal, Codon usage, Gene duplication

37 Introduction

38 Next-generation sequencing (NGS) has fostered the sequencing of many plant genomes. The 39 availability of so many genomes has allowed researchers to readily identify genes, examine 40 genetic diversity within a species, and gain insight into the evolution of genes and gene families. 41 Gene expression is regulated in part by different families of proteins known as transcription 42 factors (TFs). TFs are involved in inducing the transcription of DNA into RNA. They include 43 numerous and diverse proteins, all of which contain one or more DNA-binding motifs. The DNA- 44 binding domain enables them to bind to the promoter or repressor sequence of DNA that is 45 present either at the upstream, downstream, or within an intron region of a coding gene. Some TFs 46 bind to a DNA promoter region located near the transcription start site of a gene and help to form 47 the transcription initiation complex. Other TFs bind to regulatory enhancer sequences and 48 stimulate or repress transcription of the related genes. Regulating transcription is of paramount 49 importance to controlling gene expression and TFs enable the expression of an individual gene in 50 a unique manner, such as during different stages of development or in response to biotic or abiotic 51 stress. TFs act as a molecular switch for temporal and spatial gene regulation. A considerable 52 portion of a genome consists of genes encoding transcription factors. For example, there are at 53 least 52 TF families in Arabidopsis thaliana, and the NAC (no apical meristem (NAM) TF family 54 is one of them.

55 NAC TFs are characterised by the presence of a conserved N-terminal NAC domain comprising 56 approximately 150 amino acids and a diversified C-terminal end. The DNA binding NAC domain 57 is divided into five sub-domains designated A-E. Sub-domain A is apparently involved in the 58 formation of functional dimers, while sub-domains B and E appear to be responsible for the 59 functional divergence of NAC genes 1–4. The dimeric architecture of NAC proteins can remain 60 stable even at a concentration of 5M NaCl 4. The dimerization is established by Leu14-Thr23, 61 and Glu26-Tyr31 amino acid residues. The dimeric form is responsible for the functional unit of 62 stress-responsive SNAC1 and can modulate DNA-binding specificity. Sub-domains C and D 63 contain positively charged amino acids that bind to DNA. The crystal structure of the SNAC1 TF 64 revealed the presence of a central semi-β-barrel formed from seven twisted anti-parallel β-strands 65 with three α-helices 4. The NAC domain is most responsible for DNA binding activity that lies 66 between amino acids Val119-Ser183, Lys123-Lys126, with Lys79, Arg85, and Arg88 reside 67 within different strands of β-sheets 2,5,6. The remaining portion of the NAC domain contains a 68 loop region composed of the amino acids, Gly144-Gly149 and Lys180-Asn183, which are very 69 flexible in nature 4. The loop region of SNAC1 is quite long and different from the loop region of 70 ANAC, an abscisic-acid-responsive NAC, and could underlie the basis for different biological 71 functions. NAC TFs possesses mono or bipartite nuclear localization signals which contain a Lys 72 residue in sub-domain D 1,6–8. In addition, NAC proteins, as part of a mechanism of self- 73 regulation, also modulate the expression of several other proteins 6,9. The D subunit of a few NAC 74 TFs contain a hydrophobic negative regulatory domain (NRD), comprised of L-V-F-Y amino 75 acids, which is involved in suppressing transcriptional activity 10. For example, the NRD domain 76 can suppress the transcriptional activity of Dof, WRKY, and APETALA 2/dehydration responsive 77 elements (AP2/DRE) TFs 10.

78 Studies indicate that the diverse C-terminal domain contains a transcription regulatory region 79 (TRR) which has several group-specific motifs that can activate or repress transcription activity 80 11–14. The diverse C-terminal region imparts differences in the function of individual NAC 81 proteins by regulating the interaction of NAC TFs with diverse target proteins. Although the C- 82 terminal region of NAC TFs is diverse, it also contains group-specific conserved motifs 15. 83 Although the diverse aspects of NAC TFs have been studied, most were conducted within 84 individual plant species. A detailed comparative study of the genomic, molecular biology, and 85 evolution of NAC TFs has not been conducted. Therefore, a comprehensive analysis of NAC TFs 86 is presented in the current study.

87 Results and discussion

88 NAC transcription factors exhibit diverse genomic and biochemical features

89 Advancements in genome sequencing technology have enabled the discovery of the genomic 90 details of hundreds of plant species. The availability this genome sequence data allowed us to 91 characterize the genomic details of NAC TFs in diverse plant species. The presence of NAC TFs 92 in 160 species (18774 NAC sequences) was identified and served as the basis of the conducted 93 analyses. Comparisons of NAC sequences revealed that Brassica napus has the highest number 94 (410) of NAC TFs, while the pteridophyte plant, Marchantia polymorpha, was found to contain 95 the lowest number (9) (Table 1). On average, monocot plants contain a higher (141.20) number of 96 NAC TFs relative to dicot plants (125.56). Except for Hordeum vulgare (76), Saccharum 97 officinarum (44), and Zostera marina (62) all other monocot species possess more than one 98 hundred NAC TFs each (Table 1). Lower eukaryotic plants, bryophytes and pteridophytes also 99 possess NAC TFs. In addition, the algal species, Klebsormidium flaccidum, also contains NAC 100 TFs and this finding represents the first report of NAC TFs in algae (Table 1). A NAC TF in 101 Trifolium pratense (Tp57577_TGAC_v2_mRNA14116) was found to be the largest NAC TF, 102 comprising 3101 amino acids, while a NAC TF in Fragaria x ananassa 103 (FANhyb_icon00034378_a.1.g00001.1) was found to be the smallest NAC TF, comprising only 104 25 amino acids. Although it only contains a 25 amino acid sequence, it still encodes a NAC 105 domain. Typically, NAC TFs contain a single NAC domain located near the N-terminal region of 106 the protein. The current analysis, however, also identified NAC TFs with two NAC domains. At 107 least 77 of the 160 studied species were found to contain two NAC domains (Table 1).

108 Multiple sequence alignment revealed the presence of a conserved consensus sequence at 109 the N-terminus. The major conserved consensus sequences are P-G-F-R-F-H-P-T-D-D/E-L-I/V, 110 Y-L-x2-K, D-L-x-K-x2-P-W-x-L-P, E-W-Y-F-F, G-Y-W-K-A/T-T-G-x-D-x 1-2-I/V, G-x-K-K-x- 111 L-V-F-Y, and T-x-W-x-M-H-E-Y. Among these consensus sequences, D-D/E-L-I/V, E-W-Y-F-F, 112 G-Y-W-K, and M-H-E-Y are the conserved motifs most observed. The D-D/E-L motif is a 113 characteristic feature of the calcium-binding motifs present in the EF-hand of calcium-dependent 114 protein kinases and the presence of this motif in NAC TFs indicates that they have the potential to 115 regulate Ca2+ signalling events in cells 16. The D-D-E/E motif is located in the β’ sheet whereas 116 the Y-L-x2-K motif is in the α1a/b chain. Except for G-F-R-F-H-P-T-D-D/E-L-I/V, the conserved 117 consensus sequences contain the positively charged amino acids Lys (L) and Arg (K) that can 118 bind to negatively charged DNA. Welner et al. (2012) published the crystal structure of

119 ANAC019 and reported that Y94-W-K-A-T-G-T-D in β3, I11-K-K-A-L-V-F-Y of β4, K123-A-P-K- 120 G-T-K-T-N-W in the loop between β4 and β5, and I133-M-H-E-Y-R of β5 and Y160-K-K-Q at the 121 C-terminal end are located close to the bound DNA and are associated with DNA binding activity 122 17. They reported that Y94-W-K-A-T-G-T-D is responsible for the specific recognition of DNA 123 and binds at the major groove within DNA, whereas I11-K-K-A-L-V-F-Y, K123-A-P-K-G-T-K-T- 124 N-W, I133-M-H-E-Y-R, and Y160-K-K-Q bind to the backbone of the DNA molecule and provide 125 affinity for DNA binding activity 17. In the present analysis of 160 plant species, the identification 126 of the conserved consensus sequences G-Y-W-K-A/T-T-G-x-D-x1-2-I/V, G-x-K-K-x-L-V-F-Y, 127 and T-x-W-x-M-H-E-Y is in agreement with Welner et al (2012); suggesting that NAC TFs 128 contain conserved consensus sequences for specific DNA recognition and increasing the affinity 129 for DNA binding.

130 Hao et al., (2010) reported that the D subunit of NAC TFs contain a hydrophobic L-V-F-Y 131 amino acid motif that suppresses WRKY, Dof, and APETALA2 transcriptional regulators. This 132 suggests that NAC TFs may also function as a negative regulator of transcription 10. Our 133 sequence alignment, however, revealed that NAC TF family proteins in many different and 134 diverse plant species possess a conserved hydrophobic L-V-F-Y motif. As reported by Hao et al., 135 (2010), all of the NAC TFs have the potential to suppress the transcriptional activity of WRKY, 136 Dof, and APETALA 2/dehydration responsive element TFs; however, it is highly unlikely that an 137 organism could regulate transcriptional events in a specific and sustained manner if NAC will 138 conduct transcriptional repression.

139 The molecular weight of NAC TFs ranged from 346.46 kilodaltons (kDa) (Trifolium 140 pratense_Tp57577_TGAC_v2) to 2.94 kDa (Fragaria x 141 ananassa_FANhyb_icon00034378_a.1.g00001.1) (Figure 1). Among the studied NAC TFs, only 142 10 NAC proteins have a molecular weight (MW) more than 200 kDa and approximately 99 are 143 between 100 to 200 kDa. The MW of the majority of the NAC proteins range between 40 to 55 144 kDa (Figure 1). The Isoelectric point (pI) of the NAC proteins ranged from 11.47 145 (Brast01G304500.1.p, (Brachypodium stacei) to 3.60 (ObartAA03S_FGP19036, Oryza barthii). 146 The majority of the NAC TFs fell within a pI rage of 5-8 (Figure 2). Among the 18774 analysed 147 NAC TFs, the pI of 99 of them were ≥ 10. Approximately 69.28% of the NAC TFs had a pI that 148 was in an acidic range, whereas the remaining 30.72% had a pI within in a basic range. A protein 149 with a pH below the pI carries a net positive charge, whereas a protein with a pH above the pI 150 carries a net negative charge. The pI of a protein determines its transport, solubility, and sub- 151 cellular localization 18–20. Biomembranes, such as those surrounding the nucleus, are negatively 152 charged; as a result, positively charged (acidic pI) NAC TFs are readily attracted to the nuclear 153 membrane and subsequently transported into the nucleus to function in transcriptional regulation. 154 There are, however, approximately 30.72% NAC TFs that possess a basic Pi; suggesting that they 155 are localized in the cytosol or plasma membrane of the cell. The major role of TFs is to bind to 156 specific DNA sequences to regulate transcription. The majority of proteins have either an acidic or 157 basic pI and those with a neutral pI close to 7.4 are few because proteins tend to be insoluble, 158 unreactive, and unstable at a pH close to its pI. This is the main reason why among the 18774 159 NAC TFs analysed, only two (XP_010925972.1, Elaeis guineensis; Lus10008200, Linum 160 usitatissimum) had a pI 7.4. The existence of NAC proteins with a pI above 10 led us to speculate 161 whether these TFs function while attached to a transmembrane domain. Therefore, additional

162 analyses were conducted to determine if NAC TFs also have the potential to bind to the 163 transmembrane domain or if the NAC TFs with a basic pI remain within the cytosol.

164 NAC TFs are membrane bound

165 Transcription factors regulate diverse cellular events at transcriptional, translational, and 166 posttranslational levels. They are also involved in nuclear transport and posttranslational 167 modifications. In several cases, TFs are synthesized but remain inactive in the cytoplasm and are 168 only induced into activity through non-covalent interactions 21,22. TFs are able to remain inactive 169 through their physical association with intracellular membranes and are released by proteolytic 170 cleavage. NAC TFs are a family of proteins whose numbers are in the hundreds in the majority of 171 plant species. The fact that NAC TFs are such a large protein family, it is not surprising that NAC 172 TFs have evolved diverse functional roles. Therefore, it is plausible that NAC TFs may be 173 associated with sub-cellular organelle other than the nucleus to fulfil their diverse functional roles. 174 It is essential, however, to confirm if NAC TFs contain signalling sequences for transmembrane 175 localization. Therefore, we analysed the NAC gene sequences to determine if the signalling 176 sequences present in NAC TFs possess a transmembrane domain.

177 Results indicated that at least 2190 (8.57%) NAC TFs possess a transmembrane domain. 178 Transmembrane domains were found at both the N- and C-terminal ends of NAC proteins. In the 179 majority of the cases, however, the transmembrane domain was located towards the C-terminal 180 end. Seo et al., (2008) indicated the presence of a transmembrane domain in TFs and suggested 181 that transmembrane domain functions through two proteolytic mechanisms, commonly known as 182 regulated ubiquitin/proteasome-dependent (RUP) and regulated intramenbrane proteolysis (RIP) 183 23,24. The bZIP plant TF is present as an integral membrane protein associated with stress response 184 in the endoplasmic reticulum (ER) 25–28. Studies suggest that the majority of membrane bound TFs 185 are associated with the ER and a membrane bound TF was also found to be involved in cell 186 division 29,30. At least 10% of the TFs in Arabidopsis thaliana have been reported to be 187 transmembrane bound 30. The collective evidence clearly indicates that membrane-mediated 188 transcriptional regulation is a common stress response and that NAC TFs play a vital role in stress 189 resistance in the ER. Therefore, these membrane-bound NAC TFs can be of great importance for 190 the manipulation of stress resistance using biotechnology.

191 NAC TF contain monopartite, bipartite, non-canonical, and nuclear export signal sequences

192 The import of NAC TFs into the nucleus is mediated by nuclear membrane-bound importins and 193 exportins that form a ternary complex consisting of importin α, importin β1, and a cargo molecule. 194 Importin α serve as an adaptor molecule of importin β1 and recognises the nuclear localization 195 signal (NLS) of the cargo protein needing to be imported. Importin β1 and β2, however, also 196 recognize the NLS directly and bind to the cargo protein. Although the NLS of TFs have been 197 widely studied in the animal kingdom, their study in plants has been more restricted. Therefore, 198 the NLS of NAC TFs was examined in the current study. Results indicate that NAC TFs contain 199 diverse NLS. The NLS were found in the N- and C-terminal regions of NAC TF proteins. Some 200 NAC TFs were found to contain only one NLS whereas other contain multiple NLS. At least 3579 201 of the total NAC TFs analysed were found to contain either one or multiple NLS. More 202 specifically, 2604 NAC TFs were found to possess only one NLS at the N-terminal end of the

203 NAC protein, whereas 975 were found to possess two NLS, 254 possess three NLS, and 48 were 204 possess four NLS. The NLS were located towards the N-terminal end in the majority of NAC 205 proteins.

206 NLS motifs are rich in positively charged amino acids and bind to importin α to be 207 imported into the nucleus. The NLS motifs are classified as monopartite or bipartite. A 208 monopartite NLS contains a single cluster of positively charged amino acids and are grouped into 209 two subclasses, class-I and class-II. Class-I possesses four consecutives positively charged amino 210 acids and class-II contains three positively charged amino acids, represented by K(K/R)-x-K/R; 211 where x represents any amino acid that is present after two basic amino acids. Bipartite NLS 212 motifs contain two clusters of positively charged amino acids separated by a 10-12 amino acid 213 linker sequence. Bipartite NLS motifs are characterised by the consensus sequence K-R-P-A-A-T- 214 K-K-A-G-Q-A-K-K-K-K. In addition to monopartite and bipartite NLS motifs, importin α also 215 recognises non-canonical NLS motifs. Non-canonical NLS motifs are longer and considerably 216 variable relative to monopartite and bipartite NLS motifs and are classified as class-III and class- 217 IV NLS. Non-canonical NLS motifs are usually present in the C-terminal end and bind with 218 importin β2. Class-III and class-IV NLS motifs contain K-R-x(W/F/Y)-x2-A-F and (P/R)-x2-K-R- 219 (K/R) consensus sequences, respectively. We identified at least 1702 unique NLS consensus 220 sequences in the N-terminal region of NAC TFs. The monopartite class I NLS motifs were found 221 to contain more than four consecutive basic amino acids with the number of their consecutive 222 basic amino acids ranging from four to fourteen (K-K-K-K-K-K-K-K-K-K-K-K-K-K-K). The 223 bipartite NLS motifs contain two clusters of consecutive basic amino acids separated by up to 224 twenty-four linker amino acids (K-K-K-x3-R- x2-R- x4-K- x3-K- x3-K-x-K- x2-R-K-K).

225 The non-canonical NLS motifs contain at least six centrally-located, positively charged 226 amino acids (K-x-R-R-R-P-R-R-x2-R-K) flanked by positively charged amino acids on both sides. 227 Our analysis of the N-terminal NLS of NAC TFs, however, did not identify any NAC TFs 228 containing this consensus sequence. Instead, several new variants of this consensus sequence were 229 identified with multiple clusters of positively charged amino acids. These NLS were designated as 230 multipartite NLS motifs. A few examples of the multipartite NLS include, K-K-K-K-x7-K-K-K-

231 K-x7-K-K-K-K, K-K-K-K-x-K-x5-K-x-K-K-x7-K-K-K-K-x2-K-K-K, K-K-K-x2-K-K-x-K-x5-K- 232 x4-K-K-K-R-x-K-R-K-x-K-x4-K-K-K-R-K-K, K-K-R-x-R-K-x2-K-x-K-x2-K-K-K-x-RK-x2-K-R- 233 R-x2-K-K-K-x-R, K-K-R-x-R-K-x2-K-x-K- x2-K-K-R-x-R-K-x2-K-x-K-x2-K-K-R-x-R-K-x2-K- 234 x2-K-x-K-x-R, K-x2-K-K-K-x3-K-K-K-K-K-x-K-x8-K-x9-K-x2-K-K-R-x2-K-K-K-K-x-K, K-x2-K- 235 K-K-x3-K-x-K-K-K-x-K-K-K-x2-K-K-K-x-K, R-K-R-x-R-x-R-K-K-x2-K-x-K-K-K-R-x2-K-x2- 236 KK-x2-R-R-K-x2-K, and R-K-R-x-R-x-R-x2-K-x-K-K-K-R-x2-K-x4-K-R-x2-R-R-K-x-K-x2-R. 237 Much of the diversity of NLS motifs is associated with the sequence of the variable linker amino 238 acids. In our analysis, we removed the linker amino acid sequences, represented as x, to obtain a 239 more concise picture of NLS diversity. Removing the linker amino acids present in monopartite, 240 bipartite, and multipartite NLS motifs resulted in the identification of 97 different NLS consensus 241 sequences in the N-terminal region of NAC TFs. The unique NLS signal sequences were R-K-R- 242 R-K, K-K-K, K-R-K, K-K-R, K-R-R, R-R-R, R-K-K, R-K-R, K-K-K-K, R-K-R-K, R-R-K, R-R- 243 R-R, K-K-R-K, K-K-R-K-R, K-R-K-R, R-K-R-R-R, R-K-R-R, K-K-K-K-K, R-R-K-R, K-R-K-R- 244 R-K, R-R-K-K, R-R-R-K, K-R-R-K, K-K-R-R, R-K-R-K-R, R-R-R-R-R, K-R-K-K, K-R-R-R, K- 245 R-K-R-R-R, K-K-K-R, R-K-K-K, K-R-K-K-K, K-R-K-R-K, R-K-K-R, K-R-R-R-R, R-R-K-R-R-

246 K, K-K-K-K-R, K-K-K-R-K, K-K-R-K-K, K-K-K-K-K-K, R-K-K-R-K, R-K-R-K-K, R-K-K-K- 247 K, R-K-R-K-R-K, R-R-K-K-K, R-R-K-R-R-R, R-R-R-R-K, K-K-K-K-K-K-K, K-K-K-K-K-K-K- 248 K, K-K-K-K-R-R, K-K-R-R-R, K-K-R-R-R, R-R-K-R-K-R, R-R-R-K-K-K, R-R-R-R-R-R, R-R- 249 R-R-R-R-K, K-K-K-K-K-R, K-K-K-K-K-R-K, K-K-K-R-K-K, K-K-K-R-R-R-R-R, K-R-K-K-K- 250 K, K-R-K-R-R, K-R-R-K-R, R-K-K-K-K-R, R-K-R-K-K-K, R-K-R-R-K-R-K, R-R-K-K-K-K, R- 251 R-R-K-K, R-R-R-K-R, R-R-R-R-K-K, R-R-R-R-R-K, K-K-K-K-K-K-K-K, K-K-K-K-K-K-K-K- 252 K-K-K-K-K-K, K-K-K-K-K-K-K-K-K-K-K-K-K-K-K, K-K-K-K-K-R-R, K-K-K-K-R-K-R, K- 253 K-K-R-K-R, K-K-K-R-R-R-R, K-K-K-R-R-R-R-R-R, K-K-R-R-R-R-R-R-R-R, K-R-K-K-R, K- 254 R-K-R-K-R-K-K, K-R-R-K-K, R-K-K-K-K-K-R, R-K-K-R-K-K-R, R-K-K-R-K-R, R-K-R-K-R- 255 R, R-K-R-K-R-R-K, R-K-R-R-K, R-K-R-R-K-R, R-R-K-K-R, R-R-K-K-R-K, R-R-K-R-K, R-R- 256 R-K-R-R, R-R-R-R-K-K-K, R-R-R-R-K-R, R-R-R-R-R-R-R, and R-R-R-R-R-R-R-R. The R-K- 257 R-R-K consensus sequence was found to be present 347 times, K-K-K 297 times, K-R-K 185 258 times, K-K-R 165 times, K-R-R 153 times, R-R-R 96 times, R-K-K 95 times, R-K-R 83 times, K- 259 K-K-K 75 times, R-R-K 74 times, R-R-R-R 58 times, K-K-R-K 49 times, K-K-R-K-R 49 times, 260 and K-R-K-R 40 times. At least 27 NLS amino acid consensus sequences were only found once 261 among the 160 studied species.

262 The C-terminal end of NAC TF proteins also contain monopartite, bipartite, and 263 multipartite NLS motifs. The multipartite NLS motifs found in the C-terminal end of NAC 264 proteins were R-K-R-x-R-x-R-K-K-x4-K-x-K-K-K-R-x3-K-x3-K-K-x3-R-R-K-x2-K, R-R-R-x4-K- 265 K-x6-R-x2-R-x2-R-R-x4-R-R-R-x6-R-x2-R-R-x9-R-R-R-R-R-R-R-x2-R-R, K-K-K-x4-K-K-x-K-x5- 266 K-x4-K-K-K-R-x-K-R-K-x-K-x4-K-K-K-R-K-K, K-K-R-x4-K-x2-K-x-K-x2-K-K-R-x-R-K-x4-K- 267 x2-K-x-K-K-R-x-R-K-x4-K-x2-K-x-K-x-R, K-K-R-x-R-K-x2-K-x-K-x2-K-K-K-x-R-K-x2-K-R-R- 268 x2-K-K-K-x-R, K-K-R-x-R-K-x2-K-x-K-x2-K-K-R-x-R-K-x2-K-x-K-x2-K-K-R, R-K-R-x-R-x3-K- 269 K-R-R-x2-K-x9-K-x4-R-x-K-x2-R-x-R-R-x5-K-K-R, R-K-R-x-R-x-R-x5-K-x-K-K-K-R-x3-K-x4-K- 270 R-x2-R-R-K, R-R-x-R-R-R-x-R-R-x8-R-x6-R-R-x5-R-R-R-x-R-x5-R-x8-R-R-R-R, R-R-x-R-R-x- 271 R-x-R-R-R-x9-R-x2-R-R-K-R-K-x-R-x4-R-R-R-R-R-R-x4-R-K, R-x-R-R-R-R-x6-R-x11-R-x8-R-R-

272 x3-R-R-R-x2-R-R-x-R-x-R-x6-R-R-R-R-R-x4-R-R-x2-R, R-x-R-R-x3-K-R-R-R-x2-R-x-R-R-x-R-x- 273 R-x7-R-x3-R-R-R-x7-R-x2-R-R-R-R, R-x-R-x-R-R-R-x3-R-R-R-x3-R-x-R-x2-R-x4-R-R-R-x5-R-K- 274 x-R-x3-R-R- x13-R-R-x-K-x5-R-R-x6-K-R-R, and others. Removal of the linker amino acids 275 present in between the consecutive basic amino acids, resulted in the identification of 94 unique 276 consensus sequences. These include K-K-K, K-K-R, R-R-R, K-R-K, K-K-R-K-R, R-K-K, K-K-R- 277 K, R-K-R-K, K-K-K-K, K-R-K-R, K-R-R, R-K-R, R-R-K, R-R-R-R, K-R-K-K, R-K-R-R-K, and 278 others. The NLS consensus sequence K-K-K was identified 144 times, K-K-R 83 times, R-R-R 65 279 times, K-R-K 60 times, K-K-R-K-R 58 times, R-K-K 47 times, K-K-R-K 45 times, R-K-R-K 40 280 times, K-K-K-K 39 times, K-R-K-R 37 times, K-R-R 36 times, R-K-R 35 times, R-R-K 31 times, 281 R-R-R-R 24 times, K-R-K-K 17 times, and R-K-R-R-K 17 times. A comparison of the 97 NLS 282 consensus sequence present in N-terminal region with the 94 NLS sequences present in the C- 283 terminal region indicated that 84 NLS consensus sequences were shared between the N-terminal 284 and C-terminal regions. This indicates that there is a close relationship between the NLS 285 sequences in these two regions. An analysis of the unique NLS consensus sequence in the N-and 286 C-terminal regions indicated that 13 NLS consensus sequences were unique to the N-terminal 287 region, namely R-K-R-R-K, R-R-R-R-K, K-K-K-K-R-R, K-R-K-K-K-K, R-R-K-K-K-K, K-K-K- 288 K-K-R-R, K-K-K-R-K-R, R-K-K-R-K-K-R, R-K-R-K-R-R, R-K-R-R-K-R, R-R-K-K-R-K, R-R-

289 R-K-R-R, and R-R-R-R-K-R. Similarly, nine NLS consensus sequences were unique to the C- 290 terminal region, namely K-K-R-R-K, K-K-K-K-R-K, R-R-K-K-K-R-R-R-R-R-R-R, K-K-R-K-R- 291 K, K-R-R-R-K, R-K-K-R-K-K, R-K-K-R-R, R-R-K-R-R-R-K, and R-R-R-R. Up to six classes of 292 NLS have been reported to be associated with importin α subunit 31. To the best of our knowledge, 293 this is the first report describing such a high level of diversity and dynamism in the NLS 294 consensus sequences of NAC TFs and plant transcription factors in general. This is also the first 295 report of the presence of unique NLSs in the N-and C-terminal regions of NAC TFs.

296 Several nuclear-associated proteins contain NLS, as well as nuclear export signals (NESs). 297 Proteins that perform their function within the nucleus need to be exported out of the nucleus and 298 into the cytoplasm to undergo proteosomal degradation. Therefore, a NES is required in addition 299 to an NLS. A Ran-GTP complex binds directly to an NES and mediates the nuclear export process 300 of cargo molecules 32. NES sequences contain a hydrophobic, conserved L-V-F-Y (substitute L- 301 V/I-F-M) motif separated by variable linker amino acids at both ends 33. The presence of an L-V- 302 F-Y motif in all NAC proteins, suggests that all NAC proteins have the potential to be exported 303 out of the nucleus. Hao et al. (2010), however, reported that the hydrophobic L-V-F-Y motif 304 functions as a transcriptional repressor of WRKY, Dof, and APETALA TFs. If the L-V-F-Y motif 305 acts as a transcriptional repressor, then the transcriptional activity of these TFs would be lost; 306 resulting in an unstable genome. Therefore, we suggest that the L-V-F-Y motifs do not function as 307 a transcriptional repressor but rather as a NES.

308 NAC TFs possess a complex interactome network

309 The interacting partner of a protein can provide significant information about its potential function 310 and an entire protein-protein interactome network can greatly assist in unravelling the signalling 311 cascade of the proteins. Different cascades are interlinked in signalling systems and form intricate 312 constellations that provide information about cell response and function. Thus, the interactome 313 network of NAC TFs in A. thaliana were explored. The presence of a dynamic network was 314 revealed and a diverse set of interacting protein partners of NAC TFs were identified (Figure 3, 315 Table 2). Results indicated that NAC TFs interact with RNS1 (ribonuclease 1), ERD14 (early 316 responsive to dehydration 14), VND1 (vascular related NAC domain 1), VND7, GAI (gibberellins 317 inducible), ZF-HD1 (zinc finger homeodomain 1), TCP8 (Teosinte branched, Cycloidea, and 318 Proliferating cell nuclear antigen factor 8), TCP20, CPL1 (C-terminal domain phosphatise-like1), 319 RHA1A (ring H2 finger H1A), RHA2A, SHR (short root), PHB (phabulosa), PLT2 (plethora 2), 320 MYB59, HB23 (homeobox 23), HB30, NAC1, NAC6, NAC19, NAC32, NAC41, NAC45, 321 NAC50, NAC52, NAC76, NAC83, NAC97, NAC101, NAC105, IAA14 (auxin responsive protein 322 indole3-acetic acid), HAI1 (protein phosphatase), ABI1 (ABA insensitive 1), RVE2 (reveille), 323 PYL4 (PYR-like 4), BRM (brahma), HB52, RCD1 (radical-induced cell death 1), JMJ14 (jumonji 324 14), TPL (TOPLESS), F2P16 (TOPLESS related), TOPLESS, RING/U-box, ZF-domain (zinc 325 finger domain), SRO1 (similar to RCD 1), CUC2 (cup shaped cotyledon 2), PAS1 (pasticcino 1), 326 TI1 (defensin like 1), TSPO (tryptophan rich sensory protein), TIP2.2 (TCV-interacting protein 327 2.2), TIP3.1, T21F11.18 (TOPLESS related), LRR (leucine rich repeat), RPA2 (replicon protein 328 A2), and VR-NAC (Table 2). The interaction of NAC TF proteins with the diverse number of 329 listed proteins has been experimentally validated in A. thaliana. The interactome network includes 330 stress responsive proteins, other transcription factors, hormonal signalling proteins, protein

331 phosphatases, and defense related proteins. In addition to experimentally-validated interacting 332 proteins, bioinformatic mining indicated that NAC TFs also interact with several other proteins 333 (Table 2). Some of the identified interacting proteins were MYB, NAC, NTL (NTL2-like), 334 UBC30 (ubiquitin conjugating enzyme 30), ATM (Ataxia-Telangiectasia mutated), ATR 335 (serine/threonine kinase ATR), KNAT (knox tail), AOX1A (alternative oxidase 1A), ASG2 336 (altered seed germination 2), NYE (nonyellowing), CPL, TMO6 (target of monopteros 6), 337 RHA2A (ring H2-finger A2A), XCP (xylem cysteine peptidase), DBP (downstream auxin 338 binding), WAK5 (wall associated kinase 5), RCD, CYP71A25 (cytochrome P71A25), Stay green, 339 ERF (ethylene responsive transcription factor), PPR (pentatricopeptide), WOX (Wuschel related 340 homeobox), PPD6 (psbP-domain protein 6), MFDX (mitochondrial ferredoxin), LEA (late 341 embryogenesis abundant), IRX (irregular xylem), CESA4 (cellulose synthase A4), SCRL20 342 (SCR-like 20), PIP1-5 (aquaporin ), PUP4 (purine permease 4), XERO1 (dehydrin xero 1), SWAP 343 (suppression of white apricot), TIR-NBS, NBS-LRR, PAS, CHI (chitinase), MC5 (metacaspase 344 5), XCP (xylem cysteine peptidase), RNS, LAC (laccase), TIR-NBS (toll/interleukin receptor- 345 nucleotide binding site), NBS-LRR, DTA4 (downstream target 4), BAG6 (BCL-2 associated 346 anthogene 6), and others as well (Table 2). Some NAC TFs are co-expressed with other proteins. 347 These include DREB2A, XCP1, XCP2, ATM, ATR, MYB63, MYB69, MYB83, IRX1, AOX1A, 348 RCD1, ASG2, ERD1, TMO6, DOF6, SHR, PLT2GRP20, CYP86C4, VND7, NAC6, NAC32, 349 NAC97, NAC19, NAC102, HAI1, WRKY33, WRKY46, WRKY53 and others (Table 2). Some of 350 the NAC TFs directly interact with the interacting partner while others form complexes and 351 appear to play an indirect role. NAC TFs act as a negative regulator of ABA signalling, while 352 they induce JA/ET-associated marker genes 34.

353 The expression of several of NAC genes are either up- or down-regulated by auxin, 354 ethylene, or ABA, suggesting that NAC TFs play a role in plant hormonal signalling 35–37. One of 355 the most challenging aspects of a protein-protein interactome network is that the interaction can 356 vary depending upon the cell and its environment 38. Therefore, it is necessary to investigate the 357 dynamic interactions of proteins in different cells and environmental conditions to completely 358 understand their interacting partner and the cellular function of the TF. NAC TFs regulate ERD 359 and NCED (ABA biosynthesis) genes through a direct interaction with their promoters 39,40. NAC 360 TFs (ANAC019, ANAC055, and ANAC072) interact with ERD1 which encodes a Clp protease 361 regulatory subunit 41. The over expression of one of these three NAC TFs, however, did not 362 induce the up-regulation of ERD1 because the induction of ERD1 depends on the co-expression 363 of a zinc finger homeodomain TF, ZFHD1 41. ANAC019 and ANAC055 interact with ABI 364 (abscisic acid insensitive), and at least five MYB TFs can bind to the NAC TF promoter region 365 42,43. In this case, the NAC DNA binding domain mediates the interaction with RHA2A and 366 ZFHD1 43.

367 NAC TFs encodes chimeric proteins and contain multiple binding sites

368 NAC TFs are characterised by the presence of a DNA binding domain. Several NAC TFs, 369 however, contain more than one NAC domain. Chimeric NAC TFs have also been identified. At 370 least 45 variants of chimeric NAC TFs were identified in our analysis (Figure 4). Several of the 371 NAC TFs were also found to possess as many as three or four NAC DNA binding domains. 372 Furthermore, the NAC domains were found to be associated with PPR (pentatricopeptide), protein

373 kinase, PI3_4_kinase_3, EF-hands (elongation factor), CRM, peptidase A1, WRKY, cytochrome 374 B561, OFOF, FFO, Dna_J2, ZF_B, TIR, LRR, CS, F-box, IQ, PPC, ENT, ABC_TM1F, 375 RWP_RK, PB1, PABC, ACT, INTEGRA, RESPO, JMJC, SAM, BRX, G_TR_2, RORP, CHCH, 376 TPR, YJEF_N, HTH, HOMEO, GH16, ANK_REP_REGION, Peroxidase, LONGIN, V_SNA, 377 RECA_2, KH_TY, APAG, RRM, carrier, and a DCO domain. At least four NAC TFs from A. 378 thaliana, ten from B. napus, four from B. rapa, two from M. domestica, four from P. virgatum, 17 379 from C. sativa, eight from D. oligosanthes, eight from E. tef, and five from L. perrieri were found 380 to possess 2 NAC domains (Supplementary Table 1). NAC TFs in several other species were also 381 found to contain two NAC domains (Supplementary Table1). When two NAC domains were 382 present, both domains were located towards the N-terminal end. NAC TFs of at least three 383 species, O. rufipogon, B. stacei, and Camelina sativa were found to possess three NAC domains 384 whereas the NAC TFs in A. lyrata (gene id: 338342), C. sativa (Csa16g052260.1), and E. tef 385 (462951506) were found to possess four NAC domains (Figure 4).

386 Other chimeric domains were also identified in different regions of the NAC protein. PPR 387 domains were found in both the N-terminal or C-terminal region, a protein kinase domain was 388 found upstream to the NAC domain, and a NAC domain was found to be adjacent to a protein 389 kinase and EF-hand domain. Additionally, a protein kinase domain was found to be followed by 390 either a NAC and a CRM domain, a NAC domain was followed by a peptidase_A1 domain, a 391 NAC domain was followed by the presence of a WRKY domain, a cytochrome_B561 domain was 392 followed by either a NAC domain and a CRM domain, a DFDF and a FFD domain were followed 393 by a cytochrome B and NAC domain, a DNAJ_2 domain was followed by a NAC domain, a 394 DNAJ_2 domain was followed by a NAC and ZF_B domain, a NAC domain was followed by 395 TIR, LRR and CS domains, a NAC domain was followed by a TIR domain, an F-box domain was 396 followed by a NAC domain, an IQ domain was followed by a NAC domain, a NAC domain was 397 followed by a ZF_B domain, an EF-hand domain was followed by a NAC domain, a NAC domain 398 was followed by a PPC domain, an ENT domain was followed by a NAC domain, a NAC domain 399 was followed by an ABC_TM1F, a NAC domain was followed by CRM domain, a NAC domain 400 was followed by a RWP_RK and a PB1 domain, a NAC domain was followed by three ACT 401 domains, a NAC domain was followed by a PABC domain, a NAC domain was followed by an 402 INTEGRA domain, a RESPO domain was followed by a NAC domain, a NAC domain was 403 followed by a JMJN and a JMJC domain, a SAM domain was followed by a NAC domain, a BRX 404 domain was followed by a NAC domain, a NAC domain was followed by ZF_B, NAC and ZF_B 405 domains, an F-box and protein kinase domain was followed by a NAC domain, a NAC domain 406 was followed by a G_TR_2 domain, an RDRP domain was followed by a NAC domain, a NAC 407 domain was followed by a CHCH domain, a TPR domain followed by a NAC domain, an F-box 408 domain followed by NAC and F-box domains, a NAC domain was followed by a YJEF_N 409 domain, a NAC domain was followed by a HTH domain, a homeobox domain was followed by a 410 NAC domain, a NAC domain was followed by three GH16_2 domains, an ANK repeat region 411 was followed by a NAC domain, a NAC domain was followed by a peroxidase domain, a NAC 412 domain was followed by a LONGIN and V_SNA domain, a NAC domain was followed by 413 RECA_2 and RECA_3 domains, a KH domain was followed by a NAC domain, a NAC domain 414 was followed by a RAB domain, a JMJN domain was followed by a NAC domain, a NAC domain 415 was followed by an APAG domain, an RRM domain was followed by a NAC domain, a carrier

416 domain was followed by a NAC domain, and a NAC domain was followed by DCO domain 417 (Figure 4).

418 The presence of chimeric domains within NAC TFs is of particular interest, especially for 419 understanding why they are there and how they impact the function of a specific NAC TF. The 420 most common domains, such as PPR, TIR, WRKY, protein kinase, ZF_B, EF-hands, cytochrome 421 B, DNAJ, F-box, peroxidase, and GH16 are involved in diverse cellular processes, including 422 transcriptional regulation of plant development and stress response 44–52. The association of a TIR 423 domain with an NBS-LRR domain is an example of the association of TF domains with other 424 domains to form chimeric proteins 53. The presence of different domains with the NAC domain 425 could potentially enable the NAC domain to assist in the function of the associated domains and 426 vice versa. For example, NAC TFs could have the potential to regulate peroxidase by possessing a 427 peroxidase domain within the NAC TF, instead of regulating it separately with another TF. The 428 presence of multiple domains can enable the co-regulation of diverse functional sites within the 429 NAC TFs. The presence of chimeric TFs has been recently reported in WRKY TFs as well 54,55. 430 Therefore, the presence of chimeric domains in NAC TFs can impart a significant dynamic aspect 431 to the ability of NAC TFs to regulate gene expression.

432 In addition to the presence of multiple chimeric domains, NAC TFs were also found to contain 433 diverse active/binding motifs for several other proteins. It is possible that NAC TFs may play a 434 dual role as a transcription factor and as an enzyme. At least 404 NAC TFs were found to possess 435 other functional motifs comprising 101 unique functional sequences (Supplementary Table 2). In 436 addition to NAC domains, the other function sequences included a Fe-2S ferredoxin-type iron- 437 sulfur binding region signature, 2-oxo acid dehydrogenase acytltransferase component lipoyl 438 binding site, 4Fe-4S ferredoxin-type iron-sulfur binding domain profile, 7,8-dihydro-6- 439 hydroxymethylepterin-pyrophosphokinase signature (30), ABC transporter family signature (2), 440 adenosine and AMP deaminase signature (2), adipokinetic hormone family signature, aldehyde 441 dehydrogenase cysteine active site (4), aldehyde dehydrogenase glutamic acid active site (28), 442 aldo/keto reductase family putative active site signature (4), alkaline phosphatise active site (5), 443 aminoacyl-transfer RNA synthetase class-I signature, aminotransferase class-II pyridoxal- 444 phosphate attachment site (15), antenna complexes beta subunit signature (2), 445 ArgE/dapE/ACY1/CPG2/yscS family signature 1 (2), aspartate and glutamate racemases signature 446 1 (3), aspartokinase signature (2), ATP binding site and proton acceptor, ATP synthase alpha and 447 beta subunit signature (19), ATP dependent DNA ligase AMP-binding site (2), bacterial 448 regulatory proteins araC family signature, beta-ketoacyl synthases active site (2), C-5 cytosine- 449 specific DNA methylases active site, cadherin domain signature, carbamoyl-phosphate synthase 450 subdomain signature 2, cysteine protease inhibitor signature (19), cytochrome p450 cysteine 451 heme-iron ligand signature (7), endopeptidase Clp serine active site (3), eukaryotic and viral 452 aspartyl proteases active site (10), FGGY family of carbohydrate kinase signature 2, fumarate 453 lyases signature, GHMP kinases putative ATP-binding domain, glucoamylase active site region 454 signature, glyceraldehyde 3-phosphate dehydrogenase active site, glycoprotease family signature, 455 glycohydrolase family 5 signature, glycosyl hydrolase family 9 active site signature 2, heavy 456 metal associated domain, hemopexin domain signature (2), histone H4 signature (4), HMG-I and 457 HMG-Y DNA binding domain (A+T hook) (9), immunoglobulins and major histocompatibility 458 complex protein signature (4), inorganic pyrophosphate signature (13), iron-containing alcohol

459 dehydrogenase signature 1, legume lectins beta-chain signature (15), lipocalin signature (23), 460 mannitol dehydrogenase signature, N-6 adenine-specific DNA methylases signature (7), neutral 461 zinc metallopeptidase, zinc binding region signature, Nt-DnaJ domain signature (2), peroxidase 462 active site signature, pfkB family of carbohydrate kinases signature 1 and 2 (4), phospholipase A2 463 histidine active site (5), phosphopantetheine attachment site (17), polygalacturonase active site 464 (2), polyprenyl synthases signature, PPM-type phosphatase domain signature, prokaryotic 465 membrane lipoprotein lipid attachment site, putative AMP binding domain signature, regulator of 466 chromosome condensation (RCC1) signature 2 (2), ribosomal protein L24e signature (7), 467 ribosome binding factor A signature, rubredoxin signature (2), serine protease, subtilase family 468 aspartic acid active site (21), serine protease, trypsin family, serine active site, serine/threonine 469 protein kinase active-site signature (3), sigma-54 interaction domain ATP-binding site A 470 signature, signal peptidase I serine active site (3), signal peptidase I signature 3 (4), soybean 471 trypsin inhibitor (Kunitz) protease inhibitor family signature, SRP54-type proteins GTP-binding 472 domain signature, sugar transport proteins signature 2, synaptobrevin signature, 473 syntaxin/epimorphin family signature, TonB-dependent receptor proteins signature 1 (7), 474 translationally controlled tumor protein (TCTP) domain signature 2, Trp-Asp (WD) repeats 475 signature (12), tubulin subunit alpha, beta, and gamma signature (2), tubulin-beta mRNA 476 autoregulation signal (2), zinc carboxypeptidases, zinc binding region 2 signature (11), zinc finger 477 BED-type profile, zinc finger C2H2 type domain signature, and a zinc-containing alcohol 478 dehydrogenase signature (2) (Supplementary Table 2). This is the first study to report the presence 479 of such a diverse number of functional sites and signature motifs in NAC TFs. Although the 480 majority of the functional domains are associated with a specific function in plants, the presence 481 of a histocompatibility complex and a translationally controlled tumor protein (TCTP) sequence 482 are of particular interest. These proteins are specifically found in animal systems and the 483 histocompatibility complex is the major contributing factor regulating the binding of antigens. 484 More specifically, TCTP is a highly conserved protein that is involved in microtubule 485 stabilization, calcium binding, and apoptosis and is associated with the early growth phase of 486 tumors 56. The presence of MHC and TCTP in association with NAC domains suggests that this 487 combination may be playing a crucial role in the plant immune system and in uncontrolled cell 488 growth. The presence of diverse functional sites in NAC TFs indicates that NAC TFs are involved 489 in diverse cellular functions and metabolic pathways. This statement is supported by the large 490 number of NAC TFs that are present in plant genomes.

491 NAC TFs are involved in diverse cellular processes

492 NAC TFs are known to possess diverse chimeric domains, as a result, it is more than likely that 493 NAC TFs are also involved in the regulation of diverse cellular pathways and cellular processes. 494 To help substantiate this premise, the interactome associated with NAC TFs in A. thaliana was 495 analysed. Results indicated that NAC TFs are potentially involved in a least 289 different cellular 496 processes and pathways (Supplementary Table 3). The majority are related to cell, tissue, and 497 organ (root, stem, meristem) development, as well as signalling processes. Several NAC TFs also 498 appear to be associated with phytohormone signalling, including auxin, gibberellin, jasmonic acid, 499 and salicylic acid signalling pathways. NAC TFs were also found to be associated with pathways 500 involved in the response to bacterial, fungal, UV, heat and other biotic and abiotic stresses 501 (Supplementary Table 3). At least 202 genes in the NAC TF interactome network were found to

502 be associated with pathways related to the nucleus, 239 were associated with intracellular 503 membranes, and 241 were associated with intracellular organelles, 20 with the endoplasmic 504 reticulum, and 3 with the nuclear matrix. If the association is designated based on the description 505 of a pathway, 127 genes were found to be associated with transcription factor activity and 506 sequence-specific DNA binding, 143 with DNA binding, 146 with nucleic acid binding, 220 with 507 organic cyclic compound binding, 220 with heterocyclic compound binding, 65 with ATP 508 binding, 49 with macromolecular complex binding, 48 with chromatin binding, 35 with ADP 509 binding, 25 with sequence-specific DNA binding, 18 with transcription regulatory region binding, 510 8 with structural constituents of the cell wall, 11 with auxin transport activity, 2 with LRR 511 binding, and 2 with bHLH transcription factor binding. These data clearly indicate that NAC TFs 512 are involved in diverse cellular processes. The identification of LRR protein in the pathway 513 description of NAC TFs agrees with the presence of an LRR domain in a chimeric NAC domain 514 of NAC TFs.

515 NAC TFs are expressed in a spatiotemporal manner

516 Patterns of NAC TF gene expression were analysed in leaf and root tissues of A. thaliana 517 treated with ammonia, nitrate, or urea (Figure 5 and Figure 6). Among a total of 120 NAC TFs, 95, 518 97, and 98 were differentially expressed in leaf tissue treated with ammonia, nitrate, or urea, 519 respectively. Leaf tissues treated with ammonia, nitrate and urea exhibited 70.14, 117.11, and 520 58.35 FPKM expression values for AtNAC1 (AT1G01010.1), AtNAC4 (AT1G02230.1), and 521 AtNAC1 (AT1G01010.1), respectively. At least 46 genes in leaves exhibited expression of more 522 than one FPKM in response to ammonia, 54 in response to nitrate, and 44 in response to urea. 523 AtNAC1 was highly expressed in ammonia and urea treated leaves. At least 24, 26, and 25 NAC 524 TFs did not exhibit any expression in leaf tissues treated with ammonia, nitrate, or urea.

525 Relative to leaf tissues, the expression of NAC TFs in root tissues was more dynamic. Root 526 tissue treated with urea exhibited the highest expression of NAC TFs relative to leaves treated 527 with ammonia or nitrate (Figure 6). The number of AtNAC TFs whose expression was one or more 528 FPKM in response to ammonia, nitrate, or urea were 75, 71, and 70, respectively. AtNAC8 529 (AT5G08790.1) was highly expressed in ammonia-treated roots, whereas, AtNAC91 530 (AT5G24590.2) was highly expressed in nitrate- and urea-treated roots. Urea, ammonia and 531 nitrate (UAN) commonly serve as a source of nitrogen (N) for plants. Analysis of the levels of 532 gene expression indicate that ammonia and nitrate modulate the expression of NAC TFs more than 533 urea. A study utilizing Pinus taeda revealed that fertilization with ammonium, nitrate, or urea 534 produces different effects on growth and drought tolerance 57. Results of the current analysis 535 indicate that AtNAC8 and AtNAC91 are the major NAC TFs involved in nitrogen assimilation 536 during plant growth.

537 Codon usage in NAC TF is dynamic

538 Codon usage bias in NAC TFs of the examined species were studied. separately. Among 61 sense 539 codons, only 14 were found in the all species. These included AAG (K), ACU (R), AGA (R), 540 AGG (R), UCU (S), AUC (I), AUG (M), CAA (Q), CCU (P), GAA (E), GCU (A), GGA (G), 541 UGG (0), and UUC (F) (Table 3). The most abundant codon was UCU (S), which was found 30 542 times in in Humulus lupulus NAC TFs (Table 3). The codons CGA (R), CGC (R), CGG (R), CGU

543 (R) were absent in 127 of the 160 examined species. ACG (T), UCG (S), CAG (Q), CAC (H), 544 CCA (P), CCC (P), CCG (P), and GCG (A) were absent in 126 of the examined species. The 545 highest relative synonymous codon usage bias (RSCU) was found to be 1.35, 1.23, 1.29 for the 546 codon AAA (K) in Ocimum tenufolium, Picea sitchensis, and Ipomea trifida. Synonymous codon- 547 usage was not observed in NAC TFs. Relative codon usage is determined by dividing the ratio of 548 observed frequency of codons by the expected frequency, provided that all of the synonymous 549 codons for the same amino acids are used equally. Relative Synonymous Codon Usage (RSCU), 550 however, is not related to the usage of amino acids. An RSCU > 1 indicates the occurrence of 551 codons more frequently than expected, while an RSCU < 1 indicates that the codon occurs less 552 frequently than expected 58,59. Non-synonymous substitution in organisms is subject to natural 553 selection 60,61. Genes with lower non-synonymous selection leads to functional diversity of a gene. 554 The presence of a low level of nonsynonymous codon usage in NAC TFs indicates that they are 555 functional and have evolved from paralogous ancestors.

556 Rate of transition of NAC TFs is higher than the rate of transversion

557 Nucleotide mutation is an integral part of the evolution of a genome and leads to the acquisition of 558 required traits and the elimination of detrimental traits from the genome. It is a regular process 559 and hundreds of thousands of nucleotides have undergone addition or deletion events in the 560 evolution of a genome. The alteration or conversion of a nucleotide occurs either through a 561 transition or a transversion. A transition event involves the interchange of two-ring purines (A and 562 G) or of one-ring pyrimidines (C and T). Transversion events the exchange of a purine for a 563 pyrimidine or vice versa. The rate at which these two events occur is important to understanding 564 of the evolution of a gene. Therefore, the rate of nucleotide substitution in NAC TFs was 565 analysed. Results indicated that the rate of transition in NAC TFs is higher than the rate of 566 transversion. The substitution of adenine with guanine was found to be highest in Linum 567 usitatissimum (15.82), while the substitution of guanine to adenine was found to be the highest in 568 Lotus japonicas (19.07). The lowest rate of substitution from adenine to guanine and vice versa 569 was found in Trifolium pratense (9.73) and Amborella trichopoda (10.8), respectively (Table 4). 570 The highest rate of substitution from thiamine to cytosine and vice versa was found in 571 Klebsormidium flaccidum (7.19) and Pseudotsuga menziesii (11.59), respectively. The lowest rate 572 of substitutions from thiamine to cytosine and vice versa was found in Capsella grandiflora (2.41) 573 and Cicer arietinum (1.62), respectively (Table 4). These data make it evident that the rates of 574 transition of purine (adenine and guanine) nucleotides are higher than the rates of pyrimidines. 575 The highest rate of transversion from adenine to thiamine and vice versa was found in Capsella 576 grandiflora (12.34 for adenine to thiamine and 9.91 for thiamine to adenine) (Table 4). The rate of 577 substitution by transversion is slower relative to the rate of substitution by transition.

578 Capsella grandiflora is a close relative of Arabidopsis thaliana and is predicted to be the 579 progenitor of Capsella bursa-pastoris. Capsella grandiflora is a self-pollinating plant and is used 580 as a model organism in evolutionary studies and the change from self-incompatibility into self- 581 compatibility. The genomic consequences of the evolution of selfing, however, is poorly 582 understood. Capsella rubella, a close relative of Capsella grandiflora, that evolved self- 583 compatibility 200,000 years ago 62 also exhibits a high rate of transversion from adenine to 584 thiamine (11.19). Thus, the higher rate of transversion from adenine to thiamine in Capsella

585 grandiflora and Capsella rubella may be a possible factor in the evolution of self-pollination. 586 Higher rates of transversion were also found in Solanum pimpinellifolium (11.4) and Castanea 587 mollissima ((11.31) Chinese chestnut). Solanum pimpinellifolium is self-pollinating and exhibits 588 high levels of stress tolerance 63. Castanea mollissima has evolved over a period of time in 589 coexistence with chestnut blight and is resistant to the pathogen. This indicates that higher rates of 590 transversion from adenine to thiamine and vice versa are associated with self-pollination and 591 stress tolerance in plants. The highest rate of substitution from guanine to cytosine and vice versa 592 was found in Arachis hypogaea (11.07), and Camelina sativa (11.46), respectively (Table 4). The 593 lowest rate of substitution from adenine to thiamine and vice versa was found in Linum 594 usitatissimum (3.72) and Klebsormidium flaccidum (6.67), respectively. Notably, the highest rate 595 of substitution from thiamine to cytosine was found in Klebsormidium flaccidum and the highest 596 rate of substitution from adenine to guanine was found in Linum usitatissimum. This indicates that 597 organisms which exhibit the highest rate of transition possess the lowest rate of transversion.

598 NAC TFs evolved from orthologous ancestors

599 A phylogenetic tree of NAC TFs was constructed to understand their evolutionary relationships. A 600 model selection was conducted before constructing the phylogenetic tree using the maximum 601 likelihood statistical method. The phylogenetic tree revealed the presence of at least seven 602 phylogenetic clustered orthologous (COGs) groups originating from a common, orthologous 603 ancestor (Figure 7). Each phylogenetic cluster was further divided into two or more sub-groups. 604 A phylogenetic tree of each individual species was subsequently constructed to examine 605 duplication and loss events in NAC TFs. The phylogenetic tree of each species was independently 606 reconciled with the collective species tree. This analysis indicated that NAC TFs in all of the 607 species were duplicated and no NAC TFs were found to be lost. This suggest that NAC TFs 608 evolved from common ancestors (orthology) and underwent numerous duplication events during 609 speciation (paralogy), which gave rise to diverse gene functions in plant development and growth. 610 We also checked for the presence of potential foreign or homologous sequences (xenologs) in 611 NAC TFs. No primary xenologs, sibling donor xenologs, sibling recipient xenologs, incompatible 612 xenologs, autoxenologs, or paraxenologs were identified in NAC TFs. Although the phylogenetic 613 tree indicates the evolution NAC TFs from common ancestors, none of the NAC genes in the 614 examined species were found to have been transferred from one species to another (Table 1). 615 Previous studies of NAC TFs in six plant species also reported a high level of duplication and 616 divergent evolution 64. The expansion of TF families was associated with an increase in the 617 structural complexity of the organism 65. Previous studies reported the lineage-specific grouping 618 of transcription factors 54,64. The phylogenetic tree of NAC TFs also revealed the presence of 619 lineage-specific clustering as well. In a few cases, however, order-specific clustering of NAC TFs 620 was also observed. For example, NAC TFs in dicot species of the Brassica lineage, including A. 621 thaliana, A. halleri, B. napus, B. rapa, R. sativus, R. raphanistrum, C. rubella, A. alpine, and 622 others, grouped together. Similarly, NAC TFs in monocot plant species, including O. sativa, O. 623 nivara, B. distachyon, and others, also grouped together.

624

625

626 Conclusion

627 NAC TFs are present in higher plants, as well as in a few species of algae. The number of NAC 628 TFs per genome and their structural and functional properties increased with the complexity of the 629 organism. The algae Klebsormidium flaccidum, a charophyte, was also found to possess NAC 630 TFs; suggesting that the evolution of NAC TFs was associated with the adaptation of plant life 631 from an aquatic to a terrestrial form. The paralogous evolution of NAC TFs underlies their diverse 632 functional role in plant growth and development. Duplication events in NAC TFs were greater 633 than deletion events and the absence of any loss of NAC TFs in different plant species indicates 634 their evolution in recent times. As NAC TFs play a pivotal role within the nucleus regulating gene 635 expression, the presence of bipartite and multipartite nuclear localization signals is of particular 636 interest and provides the basis for further investigation of their functional roles.

637

638 Materials and Methods

639 Identification of NAC TFs

640 NAC genes in the studied plant species were obtained from searches in the National Centre for 641 Biotechnology Information (NCBI), Phytozome, and Plant Genome databases 66. BLASTP and 642 hidden Markov model were used to identify the NAC TFs in different species using AtNAC1 and 643 AtBAC2 as the query sequences 67. Protein and CDS sequences of each species were collected 644 and further analysed. Protein sequences of the NAC TFs were subjected to BLASTP analysis 645 against a reference database to reconfirm them as a NAC TF of the identified species. All of the 646 NAC TF protein sequences in the examined species were also subjected to ScanProsite and 647 InterPro scans to confirm the presence of a NAC domain 68,69. Sequences that were found to 648 contain a NAC domain were considered as NAC TFs. The presence of multiple NAC domains, 649 along with the presence of chimeric NAC domains, were determined through ScanProsite and 650 InterPro scans. The presence of multiple functional sites in NAC TFs were also analysed using 651 ScanProsite software.

652 Analysis of membrane attachment and nuclear localization signal sequences

653 The presence of transmembrane domains in NAC TFs of all of the examined species were 654 identified using the TMHMM server v. 2.0 70 and default parameters. Nuclear localization signal 655 sequences in NAC TFs were identified using NLStradamus software, which uses a hidden Markov 656 model for the prediction of nuclear localization signals 71. NAC TF protein sequences were 657 uploaded in FASTA format to run the program. The parameters used to run the NLS analysis 658 were; HMM state emission and transition frequencies, 2 state HMM static; prediction type Viterbi 659 and posterior, prediction cut-off 0.4; prediction display, and image and graphic.

660 Interactome analysis of NAC TFs

661 A. thaliana NAC TFs were used to examine the complex interactome network of NAC TFs. The 662 individual interaction network of each NAC TF in A. thaliana was searched in a string database 663 that contains 9.6 million proteins from 2031 organisms 72,73. The interactome network of each of

664 NAC TF were noted and the results were later used to construct the interactome network of A. 665 thaliana NAC TFs. The presented interactome network was based on an experimentally validated 666 network, co-expressed network, and a mined network. These outputs were used to construct the 667 interactome network. The NAC TFs used to construct the interactome network were subjected to 668 GO (gene ontology) and cellular process analyses.

669 Gene expression analysis

670 Differential gene expression of NAC TFs was analysed to elucidate their role in growth, 671 development, and nitrogen assimilation. A. thaliana NAC TFs were used to examine differential 672 gene expression. Transcriptome data from A. thaliana treated with ammonia, nitrate, and urea 673 were utilized from the PhytoMine database in Phytozome. The expression pattern of NAC TFs for 674 leaf and root tissues in the treated A. thaliana plants were analysed separately. The expression was 675 measured in fragments per kilobase of exon per million fragments mapped (FPKM). Transcripts 676 with a zero value were discarded from the study.

677 Construction of a phylogenetic tree

678 Two approaches were used to construct phylogenetic trees. In the first approach, a phylogenetic 679 tree was constructed using the NAC TFs of individual species. In the second approach, the NAC 680 TFs of all of the examined species were used to construct a phylogenetic tree. The phylogenetic 681 tree for individual species was constructed to determine the deletion and duplication events in 682 NAC TFs within individual species. Prior to construction of the phylogenetic trees, a model 683 selection was carried out in MEGA6 software. The following parameters were used in the model, 684 analysis, model selection; tree to use, automatic (neighbor joining), statistical method, maximum 685 likelihood; substitution type, nucleotides; gaps/missing data treatment, partial deletion; site 686 coverage cut-off (%), 95; codons included, 1st+2nd+3rd+non-coding. Based on the lowest BIC 687 values of model selection, phylogenetic trees of NAC TFs were carried out using the neighbor 688 joining method, a GTR statistical model, and 1000 bootstrap replicates.

689 Analysis of transition and transversion rates

690 Transition and transversion rates in NAC TFs within individual species were analysed using 691 MEGA6 software. The converted MEGA file format of individual species was used to determine 692 the rate of transition and transversion. The following statistical parameters were used to study the 693 transition/transversion rate: estimate transition/transversion bias; maximum composite likelihood 694 estimates of the pattern of nucleotide substitution; substitution type, nucleotides; model/method, 695 Tamura-Nei; gaps/missing data treatment, pairwise deletion; codon position, 1st, 2nd, 3rd, and non- 696 coding sites.

697 Analysis of gene deletion and duplication

698 Prior to the analysis of deletion and duplication events in NAC TFs, a species tree was constructed 699 in the NCBI taxonomy browser 700 (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi). All of the studied species 701 were used to construct the species tree. The resulting phylogenetic trees of individual species in a 702 nwk file format were uploaded in Notung2.9 software as a gene tree and reconciled as a gene tree

703 with the species tree to obtain duplicated and deleted genes. Deletion and duplication events were 704 analysed in all of the studied species individually.

705 Data availability

706 All the data used during this study was taken from publicly available genomic databases and 707 details are mentioned in the materials and methods section.

708 Competing of interest

709 Authors don’t have any competing of interest to declare.

710

711

712

713

714

715

716

717

718

719

720

721

722

723

724

725

726

727

728

729

730

731

732 References

733 1. Puranik, S., Sahu, P. P., Srivastava, P. S. & Prasad, M. NAC proteins: Regulation and role 734 in stress tolerance. Trends Plant Sci. 17, 369–381 (2012).

735 2. Ernst, H. A., Nina Olsen, A., Skriver, K., Larsen, S. & Lo Leggio, L. Structure of the 736 conserved domain of ANAC, a member of the NAC family of transcription factors. EMBO 737 Rep. 5, 297–303 (2004).

738 3. Jensen, M. et al. The Arabidopsis thaliana NAC transcription factor family: structure- 739 function relationships and determinants of ANAC019 stress signalling. Biochem. J. 426, 740 183–196 (2009).

741 4. Chen, Q., Wang, Q., Xiong, L. & Lou, Z. A structural view of the conserved domain of rice 742 stress-responsive NAC1. Protein Cell 2, 55–63 (2011).

743 5. Duval, M., Hsieh, T.-F., Kim, S. Y. & Thomas, T. L. Molecular characterization of 744 AtNAM: a member of theArabidopsis NAC domain superfamily. Plant Mol. Biol. 50, 237– 745 248 (2002).

746 6. Olsen, A. N., Ernst, H. A., Leggio, L. Lo & Skriver, K. NAC transcription factors: 747 structurally distinct, functionally diverse. Trends Plant Sci. 10, 79–87 (2005).

748 7. Le, D. T. et al. Genome-Wide Survey and Expression Analysis of the Plant-Specific NAC 749 Transcription Factor Family in Soybean During Development and Dehydration Stress. DNA 750 Res. An Int. J. Rapid Publ. Reports Genes Genomes 18, 263–276 (2011).

751 8. Olsen, A. N., Ernst, H. A., Leggio, L. Lo & Skriver, K. DNA-binding specificity and 752 molecular functions of NAC transcription factors. Plant Sci. 169, 785–797 (2005).

753 9. Greve, K., La Cour, T., Jensen, M. K., Poulsen, F. M. & Skriver, K. Interactions between 754 plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 755 molecular specificity and cellular localization. Biochem. J. 371, 97–108 (2003).

756 10. Hao, Y.-J. et al. Plant NAC-type transcription factor proteins contain a NARD domain for 757 repression of transcriptional activation. Planta 232, 1033–1043 (2010).

758 11. Fang, Y., You, J., Xie, K., Xie, W. & Xiong, L. Systematic sequence analysis and 759 identification of tissue-specific or stress-responsive genes of NAC transcription factor 760 family in rice. Mol. Genet. Genomics 280, 547–563 (2008).

761 12. Yamaguchi, M. et al. VND-INTERACTING2, a NAC Domain Transcription Factor, 762 Negatively Regulates Xylem Vessel Formation in Arabidopsis. Plant Cell 22, 1249–1263 763 (2010).

764 13. Ho, S. K. et al. Identification of a calmodulin-binding NAC protein as a transcriptional 765 repressor in Arabidopsis. J. Biol. Chem. 282, 36292–36302 (2007).

766 14. Christian, D. et al. The transcription factor ATAF2 represses the expression of 767 pathogenesis-related genes in Arabidopsis. Plant J. 43, 745–757 (2005).

768 15. Shen, H., Yin, Y., Chen, F., Xu, Y. & Dixon, R. A. A Bioinformatic Analysis of NAC 769 Genes for Plant Cell Wall Development in Relation to Lignocellulosic Bioenergy

770 Production. BioEnergy Res. 2, 217 (2009).

771 16. Mohanta, T., Mohanta, N., Mohanta, Y. & Bae, H. Genome-Wide Identification of Calcium 772 Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of Novel D-x-D 773 and D-E-L Motifs in EF-Hand Domain. Front. Plant Sci. 6, 1146 (2015).

774 17. Welner, D. H. et al. DNA binding by the plant-specific NAC transcription factors in crystal 775 and solution: a firm link to WRKY and GCM transcription factors. Biochem. J. 444, 395– 776 404 (2012).

777 18. Shaw, K. L., Grimsley, G. R., Yakovlev, G. I., Makarov, A. A. & Pace, C. N. The effect of 778 net charge on the solubility, activity, and stability of ribonuclease Sa. Protein Sci. 10, 779 1206–1215 (2001).

780 19. Pergande, M. R. & Cologna, S. M. Isoelectric Point Separations of Peptides and Proteins. 781 Proteomes 5, 4 (2017).

782 20. Cunningham, J. et al. Intracellular Electric Field and pH Optimize Protein Localization and 783 Movement. PLoS One 7, e36894 (2012).

784 21. Hoppe, T., Rape, M. & Jentsch, S. Membrane-bound transcription factors: regulated release 785 by RIP or RUP. Curr. Opin. Cell Biol. 13, 344–348 (2001).

786 22. Vik, Å. & Rine, J. Membrane biology: Membrane-regulated transcription. Curr. Biol. 10, 787 R869–R871 (2000).

788 23. Hoppe, T. et al. Activation of a Membrane-Bound Transcription Factor by Regulated 789 Ubiquitin/Proteasome-Dependent Processing. Cell 102, 577–586 (2000).

790 24. Seo, P. J., Kim, S. G. & Park, C. M. Membrane-bound transcription factors in plants. 791 Trends Plant Sci. 13, 550–556 (2008).

792 25. Shen, J., Chen, X., Hendershot, L. & Prywes, R. ER Stress Regulation of ATF6 793 Localization by Dissociation of BiP/GRP78 Binding and Unmasking of Golgi Localization 794 Signals. Dev. Cell 3, 99–111 (2002).

795 26. Liu, J.-X., Srivastava, R., Che, P. & Howell, S. H. An Endoplasmic Reticulum Stress 796 Response in Arabidopsis Is Mediated by Proteolytic Processing and Nuclear Relocation of 797 a Membrane-Associated Transcription Factor, bZIP28. Plant Cell 19, 4111–4119 (2007).

798 27. Liu, J.-X., Srivastava, R., Che, P. & Howell, S. H. Salt stress responses in Arabidopsis 799 utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. 800 Plant J. 51, 897–909 (2007).

801 28. Iwata, Y. & Koizumi, N. An Arabidopsis transcription factor, AtbZIP60, regulates the 802 endoplasmic reticulum stress response in a manner unique to plants. Proc. Natl. Acad. Sci. 803 U. S. A. 102, 5280–5285 (2005).

804 29. Kim, Y.-S. et al. A Membrane-Bound NAC Transcription Factor Regulates Cell Division 805 in Arabidopsis. Plant Cell 18, 3132–3144 (2006).

806 30. Kim, S.-Y. et al. Exploring membrane-associated NAC transcription factors in 807 Arabidopsis: implications for membrane biology in genome regulation. Nucleic Acids Res. 808 35, 203–213 (2007).

809 31. Kosugi, S. et al. Six classes of nuclear localization signals specific to different binding 810 grooves of importin α. J. Biol. Chem. 284, 478–485 (2009).

811 32. Mattaj, I. W. & Englmeier, L. Nucleocytoplasmic Transport: The Soluble Phase. Annu. 812 Rev. Biochem. 67, 265–306 (1998).

813 33. Kosugi, S., Hasebe, M., Tomita, M. & Yanagawa, H. Nuclear export signal consensus 814 sequences defined using a localization-based yeast selection system. Traffic 9, 2053–2062 815 (2008).

816 34. Jensen, M. et al. Transcriptional regulation by an NAC (NAM–ATAF1,2–CUC2) 817 transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria 818 graminis f. sp. hordei in Arabidopsis. Plant J. 56, 867–880 (2008).

819 35. Xie, Q., Frugis, G., Colgan, D. & Chua, N.-H. Arabidopsis NAC1 transduces auxin signal 820 downstream of TIR1 to promote lateral root development. Genes Dev. 14, 3024–3036 821 (2000).

822 36. He, X. et al. AtNAC2, a transcription factor downstream of ethylene and auxin signaling 823 pathways, is involved in salt stress response and lateral root development. Plant J. 44, 903– 824 916 (2005).

825 37. Sperotto, R. A. et al. Identification of up-regulated genes in flag leaves during rice grain 826 filling and characterization of OsNAC5, a new ABA-dependent transcription factor. Planta 827 230, 985–1002 (2009).

828 38. Bonetta, L. Interactome under construction. Nature 468, 851 (2010).

829 39. Tran, L.-S. P. et al. Isolation and Functional Analysis of Arabidopsis Stress-Inducible NAC 830 Transcription Factors That Bind to a Drought-Responsive <em>cis</em>- 831 Element in the <em>early responsive to dehydration stress 1</em> Promoter. 832 Plant Cell 16, 2481 LP-2498 (2004).

833 40. Jensen, M. K. et al. ATAF1 transcription factor directly regulates abscisic acid biosynthetic 834 gene NCED3 in Arabidopsis thaliana. FEBS Open Bio 3, 321–327 (2013).

835 41. Kazuo, N., Tomohiro, K., Kazuko, Y.-S. & Kazuo, S. A nuclear gene, erd1, encoding a 836 chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water 837 stress but also developmentally up-regulated during senescence in Arabidopsis thaliana. 838 Plant J. 12, 851–861 (2003).

839 42. Reeves, W. M., Lynch, T. J., Mobin, R. & Finkelstein, R. R. Direct targets of the 840 transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 841 and several bZIP ABA response factors. Plant Mol. Biol. 75, 347–363 (2011).

842 43. Jensen, M. K. & Skriver, K. NAC transcription factor gene regulatory and protein-protein 843 interaction networks in plant stress responses and senescence. IUBMB Life 66, 156–166 844 (2014).

845 44. Jingjing, J. et al. WRKY transcription factors in plant responses to stresses. J. Integr. Plant 846 Biol. 59, 86–101 (2016).

847 45. Pandey, S. P. & Somssich, I. E. The Role of WRKY Transcription Factors in Plant 848 Immunity. Plant Physiol. 150, 1648–1655 (2009).

849 46. Saha, D., Prasad, A. M. & Srinivasan, R. Pentatricopeptide repeat proteins and their 850 emerging roles in plants. Plant Physiol. Biochem. 45, 521–534 (2007).

851 47. Barkan, A. & Small, I. Pentatricopeptide Repeat Proteins in Plants. Annu. Rev. Plant Biol. 852 65, 415–442 (2014).

853 48. Nandety, R. S. et al. The role of TIR-NBS and TIR-X proteins in plant basal defense 854 responses. Plant Physiol. 162, 1459–72 (2013).

855 49. Wan, H. et al. Analysis of TIR- and non-TIR-NBS-LRR disease resistance gene analogous 856 in pepper: characterization, genetic variation, functional divergence and expression 857 patterns. BMC Genomics 13, 502 (2012).

858 50. Mohanta, T. K. et al. Ginkgo biloba responds to herbivory by activating early signaling and 859 direct defenses. PLoS One 7, e32822 (2012).

860 51. Mohanta, T. K., Arora, P. K., Mohanta, N., Parida, P. & Bae, H. Identification of new 861 members of the MAPK gene family in plants shows diverse conserved domains and novel 862 activation loop variants. BMC Genomics 16, (2015).

863 52. Mohanta, T. K., Mohanta, N., Mohanta, Y. K. & Bae, H. Genome-Wide Identification of 864 Calcium Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of 865 Novel D-x-D and D-E-L Motifs in EF-Hand Domain. Front. Plant Sci. 6, 1146 (2015).

866 53. Zhang, X. et al. Multiple functional self-association interfaces in plant TIR domains. Proc. 867 Natl. Acad. Sci. 114, E2046 LP-E2052 (2017).

868 54. Mohanta, T. K., Park, Y.-H. & Bae, H. Novel Genomic and Evolutionary Insight of WRKY 869 Transcription Factors in Plant Lineage. Sci. Rep. 6, (2016).

870 55. Rinerson, C. I., Rabara, R. C., Tripathi, P., Shen, Q. J. & Rushton, P. J. The evolution of 871 WRKY transcription factors. BMC Plant Biol. 15, 1–18 (2015).

872 56. Holger, T., Mario, B., Angela, S. & Bernd-Joachim, T. Expression of the gene and 873 processed pseudogenes encoding the human and rabbit translationally controlled tumour 874 protein (TCTP). Eur. J. Biochem. 267, 5473–5481 (2003).

875 57. Faustino, L. I., Moretti, A. P. & Graciano, C. Fertilization with urea, ammonium and nitrate 876 produce different effects on growth, hydraulic traits and drought tolerance in Pinus taeda 877 seedlings. Tree Physiol. 35, 1062–1074 (2015).

878 58. Kirkpatrick, C. L. et al. Growth control switch by a DNA-damage-inducible toxin– 879 antitoxin system in Caulobacter crescentus. Nat. Microbiol. 1, 16008 (2016).

880 59. Xu, X., Liu, Q., Fan, L., Cui, X. & Zhou, X. Analysis of synonymous codon usage and 881 evolution of begomoviruses . J. Zhejiang Univ. Sci. B 9, 667–674 (2008).

882 60. Hu, T. & Banzhaf, W. Nonsynonymous to Synonymous Substitution Ratio k a / k s : 883 Measurement for Rate of Evolution in Evolutionary Computation. in PPSNX, LNCS 5199 884 448–457 (2008).

885 61. Tomoko, O. Synonymous and nonsynonymous substitutions in mammalian genes and the 886 nearly neutral theory. J. Mol. Evol. 40, 56–63 (1995).

887 62. Slotte, T. et al. The Capsella rubella genome and the genomic consequences of rapid

888 mating system evolution. Nat. Genet. 45, 831–5 (2013).

889 63. Bolger, A. et al. The genome of the stress-tolerant wild tomato species Solanum pennellii. 890 Nat. Genet. 46, 1034 (2014).

891 64. Jin, X. et al. Divergent Evolutionary Patterns of NAC Transcription Factors Are Associated 892 with Diversification and Gene Duplications in Angiosperm. Front. Plant Sci. 8, 1156 893 (2017).

894 65. Schmitz, J. F., Zimmer, F. & Bornberg-Bauer, E. Mechanisms of transcription factor 895 evolution in Metazoa. Nucleic Acids Res. 44, 6287–6297 (2016).

896 66. Goodstein, D. M. et al. Phytozome: a comparative platform for green plant genomics. 897 Nucleic Acids Res. 40, D1178-86 (2012).

898 67. Boratyn, G. M. et al. Domain enhanced lookup time accelerated BLAST. Biol. Direct 7, 12 899 (2012).

900 68. Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 901 30, 1236–1240 (2014).

902 69. de Castro, E. et al. ScanProsite: detection of PROSITE signature matches and ProRule- 903 associated functional and structural residues in proteins. Nucleic Acids Res. 34, W362-5 904 (2006).

905 70. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. L. Predicting transmembrane 906 protein topology with a hidden markov model: application to complete genomes11Edited 907 by F. Cohen. J. Mol. Biol. 305, 567–580 (2001).

908 71. Nguyen Ba, A., Pogoutse, A., Provart, N. & Moses, A. NLStradamus: a simple Hidden 909 Markov Model for nuclear localization signal prediction. BMC Bioinformatics 10, 202 910 (2009).

911 72. Szklarczyk, D. et al. The STRING database in 2017: quality-controlled protein–protein 912 association networks, made broadly accessible. Nucleic Acids Res. 45, D362–D368 (2017).

913 73. von Mering, C. et al. STRING: a database of predicted functional associations between 914 proteins. Nucleic Acids Res. 31, 258–261 (2003).

915

916

917

918

919

920

921

922

923 Author contributions

924 TKM: conceived the idea, performed the experiments and analysis, drafted and revised the 925 manuscript, AK: performed the analysis, DY: revised the manuscript, AH: drafted and revised the 926 manuscript, BT: revised the manuscript, ALK: analysed the data and revised the manuscript, EFA: 927 revised the manuscript, AAH: revised the manuscript.

928

929

930

931

932

933

934

935

936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951 Figure legends

952 Figure 1

953 The distribution of the molecular weight of NAC TFs. The molecular weight of NAC TFs ranged 954 from 2.94 kDa (Fragaria x ananassa, FANhyb_icon00034378_a.1.g00001.1) to 346.46 kDa 955 (Trifolium pratense, Tp57577_TGAC_v2_mRNA14116). The average molecular weight of NAC 956 TFs was 38.72 kDa. In total, 17158 NAC TFs were utilized in the analysis of molecular weight. 957 The analysis was conducted using a protein isoelectric point calculator (http://isoelectric.org/).

958 Figure 2

959 The distribution of the isoelectric point of NAC TFs. The isoelectric point of NAC TFs ranged 960 from pI 3.78 (OB07G17140.1, Oryza brachyantha) to pI 11.47 (Sevir.3G242500, Setaria viridis). 961 The average isoelectric point of NAC TFs was 6.38. A total of 17158 NAC TFs were utilized in 962 the analysis of the pI of NAC TFs. The analysis of pI was conducted using a protein isoelectric 963 point calculator (http://isoelectric.org/).

964 Figure 3

965 Interactome network of NAC TFs. The interactome network of NAC TF reflects a diverse 966 complex of interacting proteins. The NAC TFs of A. thaliana were utilized in the interactome 967 network analysis. The interactome map of A. thaliana was determined using the string database 968 (https://string-db.org).

969 Figure 4

970 Chimeric NAC domains. NAC TFs possess chimeric NAC domains with at least 34 diverse 971 chimeric NAC domains identified in the studied species. The identification of chimeric NAC 972 domain sequences was determined using the ScanProsite and InterProScan server.

973 Figure 5

974 Chimeric NAC domains NAC TFs possess chimeric NAC domains with at least 21 diverse 975 chimeric NAC domains identified in the studied species. The identification of chimeric NAC 976 domain sequences was determined using the ScanProsite and InterProScan server.

977 Figure 6

978 Differential expression of NAC TFs in leaves of A. thaliana plants treated with ammonia, nitrate, 979 and urea. The expression of A. thaliana NAC TFs was analysed to determine their response to 980 different sources of nitrogen. Expression data were obtained from the PhytoMine database in 981 Phytozome and presented as FPKM (Fragments per Kilobase of transcripts per million mapped 982 reads).

983 Figure 7

984 Differential expression of NAC TFs in roots of A. thaliana plants treated with ammonia, nitrate, 985 and urea. The expression of A. thaliana NAC TFs was analysed to determine their response to

986 different sources of nitrogen. Expression data were obtained from the PhytoMine database in 987 Phytozome and presented as FPKM (Fragments per Kilobase of transcripts per million mapped 988 reads).

989 Figure 8

990 Phylogenetic tree of NAC TFs. A phylogenetic tree of NAC TF reveals the presence of seven 991 clustered orthologous groups (COGs). Each group also possesses two or more sub-groups. The 992 phylogenetic tree shows lineage (monocot/dicot) specific grouping of NAC TFs. The phylogenetic 993 tree was constructed using the neighbor joining method with 1000 bootstrap replicates.

994

995

996

997

998

999

1000

1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015 Table 1. Genomic details of NAC TFs of plants genes ofparalogous No. No. ofgenestransfer No. chimeric NAC TFs NACchimeric

Sl. Name of the species oforthologous No. No. ofconditional No. Total No. of NAC No.of Total No. ofduplicated No. domain NAC TFdomain duplicated genes duplicated No. of lost genesoflost No.

No ofdouble No. No. ofNovel No.

genes genes TFs

Monocots 1 Aegilops tauschii 4 117 114 0 0 0 114 0 2 Brachypodium distachyon 2 1 137 135 0 0 0 135 0 3 Brachypodium stacei 1 1 128 127 0 0 0 127 0 4 Hordeum vulgare 76 76 0 0 0 76 0 5 Leersia perrieri 5 2 163 162 0 0 0 162 0 6 Oropetium thomaeum 1 118 103 0 0 0 103 0 7 Oryza barthii 4 134 138 0 0 0 138 0 8 Oryza brachyantha 1 1 118 110 0 0 0 110 0 9 Oryza glaberrima 1 116 110 0 0 0 110 0 10 Oryza glumipatula 2 140 139 0 0 0 139 0 11 Oryza longistaminata 1 6 125 98 0 0 0 98 0 12 Oryza meridionalis 2 2 127 123 0 0 0 123 0 13 Oryza nivara 4 1 146 130 0 0 0 130 0 14 Oryza punctata 6 1 135 133 0 0 0 133 0 15 Oryza rufipogon 4 3 136 129 0 0 0 129 0 16 Oryza sativa subsp. indica 1 3 157 156 0 0 0 156 0 17 Oryza sativa subsp. japonica 1 139 138 0 0 0 138 0 18 Panicum hallii 3 6 139 126 0 0 0 126 0 19 Panicum virgatum 9 6 310 309 0 0 0 309 0 20 Phoenix dactylifera 3 1 124 123 0 0 0 123 0 21 Phyllostachys edulis 125 124 0 0 0 124 0 22 Phyllostachys heterocycla 2 2 125 124 0 0 0 124 0 23 Saccharum officinarum 44 33 0 0 0 33 0 24 Setaria italica 4 139 134 0 0 0 134 0 25 Setaria viridis 1 135 118 0 0 0 118 0 26 Sorghum bicolor 1 141 134 0 0 0 134 0 27 Spirodela polyrhiza 55 48 0 0 0 48 0 28 Triticum aestivum 2 2 263 209 0 0 0 209 0 29 Triticum urartu 1 103 74 0 0 0 74 0 30 Zea mays 1 1 130 119 0 0 0 119 0 31 Zostera marina 1 62 55 0 0 0 55 0 32 Zoysia japonica 4 176 160 0 0 0 160 0 33 Zoysia matrella 1 3 313 230 0 0 0 230 0 34 Zoysia pacifica 1 2 205 183 0 0 0 183 0 Dicots 35 Actinidia chinensis 1 5 167 166 0 0 0 166 0 36 Aethionema arabicum 3 85 84 0 0 0 84 0

27 bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

37 Amaranthus hypochondriacus 1 44 37 0 0 0 37 0 38 Amborella trichopoda 46 45 0 0 0 45 0 39 Ananas comosus 1 73 72 0 0 0 72 0 40 Aquilegia coerulea 80 79 0 0 0 79 0 41 Arabidopsis halleri 2 94 93 0 0 0 93 0 42 Arabidopsis lyrata 4 1 122 121 0 0 0 121 0 43 Arabidopsis thaliana 5 113 112 0 0 0 112 0 44 Arabis alpina 1 82 81 0 0 0 81 0 45 Arachis duranensis 82 81 0 0 0 81 0 46 Arachis hypogaea 32 31 0 0 0 31 0 47 Arachis ipaensis 83 81 0 0 0 81 0 48 Artemisia annua 28 27 0 0 0 27 0 49 Azadirachta indica 183 182 0 0 0 182 0 50 Beta vulgaris 53 52 0 0 0 52 0 51 Boechera stricta 2 123 122 0 0 0 122 0 52 Brassica napus 10 7 410 409 0 0 0 409 0 53 Brassica oleracea 4 3 271 270 0 0 0 270 0 54 Brassica rapa 4 2 256 255 0 0 0 255 0 55 Cajanus cajan 96 95 0 0 0 95 0 56 Camelina sativa 17 3 341 330 0 0 0 330 0 57 Cannabis sativa 58 57 0 0 0 57 0 58 Capsella grandiflora 2 95 94 0 0 0 94 0 59 Capsella rubella 5 119 118 0 0 0 118 0 60 Capsicum annum 96 95 0 0 0 95 0 61 Carica papaya 82 81 0 0 0 81 0 62 Castanea mollissima 4 91 78 0 0 0 78 0 63 Catharanthus roseus 2 121 120 0 0 0 120 0 64 Chenopodium quinoa 1 96 95 0 0 0 95 0 65 Cicer arietinum 96 95 0 0 0 95 0 66 Citrullus lanatus 80 79 0 0 0 79 0 67 Citrus clementina 129 128 0 0 0 128 0 68 Citrus sinensis 2 145 143 0 0 0 143 0 69 Coffea canephora 63 62 0 0 0 62 0 70 Cucumis melo 92 91 0 0 0 91 0 71 Cuccumis sativus 83 80 0 0 0 80 0 72 Daucus carota 2 96 95 0 0 0 95 0 73 Dianthus caryophyllus 79 77 0 0 0 77 0 74 Dichanthelium oligosanthes 8 2 131 100 0 0 0 100 0 75 Dorcoceras hygrometricum 2 83 76 0 0 0 76 0 76 Elaeis guineensis 2 1 170 167 0 0 0 167 0 77 Eragrostis tef 8 3 172 165 0 0 0 165 0 78 Eucalyptus camaldulensis 200 124 0 0 0 124 0 79 Eucalyptus grandis 164 150 0 0 0 150 0 80 Eutrema salsugineum 2 122 104 0 0 0 104 0 81 Fragaria vesca 3 6 127 123 0 0 0 123 0 82 Fragaria x ananassa 2 1 98 97 0 0 0 97 0 83 Genlisea aurea 1 45 42 0 0 0 42 0 84 Glycine max 180 175 0 0 0 175 0 85 Glycine soja 1 173 166 0 0 0 166 0

28 bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

86 Gossypium arboreum 150 146 0 0 0 146 0 87 Gossypium hirsutum 1 2 306 296 0 0 0 296 0 88 Gossypium raimondii 153 145 0 0 0 145 0 89 Helianthus annuus 21 20 0 0 0 20 0 90 Humulus lupulus 74 68 0 0 0 68 0 91 Ipomoea trifida 1 2 131 123 0 0 0 123 0 92 Jatropha curcas 1 97 93 0 0 0 93 0 93 Juglans regia 3 92 81 0 0 0 81 0 94 Kalanchoe laxiflora 166 165 0 0 0 165 0 95 Kalanchoe marnieriana 179 178 0 0 0 178 0 96 Lactuca sativa 54 52 0 0 0 52 0 97 Linum usitatissimum 1 1 191 187 0 0 0 187 0 98 Lotus japonicus 2 98 92 0 0 0 92 0 99 Malus domestica 2 9 253 232 0 0 0 232 0 100 Manihot esculenta 130 128 0 0 0 128 0 101 Medicago truncatula 1 97 90 0 0 0 90 0 102 Mimulus guttatus 114 113 0 0 0 113 0 103 Morus notabilis 2 78 77 0 0 0 77 0 104 Musa acuminata 1 1 170 164 0 0 0 164 0 105 Nelumbo nucifera 88 79 0 0 0 79 0 106 Nicotiana benthamiana 2 2 227 185 0 0 0 185 0 107 Nicotiana sylvestris 156 149 0 0 0 149 0 108 Nicotiana tabacum 280 279 0 0 0 279 0 109 Nicotiana tomentosiformis 172 162 0 0 0 162 0 110 Ocimum tenuiflorum 2 1 110 82 0 0 0 82 0 111 Petunia axillaris 3 131 108 0 0 0 108 0 112 Petunia inflata 157 147 0 0 0 147 0 113 Phaseolus vulgaris 85 84 0 0 0 84 0 114 Populus euphratica 2 3 155 149 0 0 0 149 0 115 Populus trichocarpa 1 169 149 0 0 0 149 0 116 Prunus mume 1 129 128 0 0 0 128 0 117 Prunus persica 1 1 115 114 0 0 0 114 0 118 Pyrus bretschneideri 1 5 185 183 0 0 0 183 0 119 Raphanus raphanistrum 4 3 207 206 0 0 0 206 0 120 Raphanus sativus 5 1 217 197 0 0 0 197 0 121 Ricinus communis 95 87 0 0 0 87 0 122 Salix purpurea 175 152 0 0 0 152 0 123 Salvia miltiorrhiza 1 2 87 81 0 0 0 81 0 124 Sesamum indicum 105 104 0 0 0 104 0 125 Sisymbrium irio 2 2 121 118 0 0 0 118 0 126 Solanum lycopersicum 101 94 0 0 0 94 0 127 Solanum melongena 1 3 95 85 0 0 0 85 0 128 Solanum pennellii 2 102 98 0 0 0 98 0 129 Solanum pimpinellifolium 97 90 0 0 0 90 0 130 Solanum tuberosum 1 129 115 0 0 0 115 0 131 Spinacia oleracea 45 43 0 0 0 43 0 132 Tarenaya hassleriana 1 178 177 0 0 0 177 0 133 Thellungiella halophila 2 122 121 0 0 0 121 0 134 Thellungiella parvula 1 92 91 0 0 0 91 0

135 Theobroma cacao 132 131 0 0 0 131 0 136 Trifolium pratense 2 2 97 76 0 0 0 76 0 137 Utricularia gibba 1 74 73 0 0 0 73 0 138 Vigna angularis 98 97 0 0 0 97 0 139 Vigna radiata 2 82 81 0 0 0 81 0 140 Vigna unguiculata 20 19 0 0 0 19 0 141 Ziziphus jujuba 101 100 0 0 0 100 0 142 Vitis vinifera 1 70 79 0 0 0 79 0 Gymnosperms 143 Picea abies 1 100 73 0 0 0 73 0 144 Picea glauca 32 31 0 0 0 31 0 145 Picea sitchensis 16 15 0 0 0 15 0 146 Pinus taeda 31 27 0 0 0 27 0 147 Pseudotsuga menziesii 5 3 196 195 0 0 0 195 0 Pteridophyte 148 Selaginella moellendorffii 22 21 0 0 0 21 0 Bryophytes 149 Marchantia polymorpha 9 150 Physcomitrella patens 33 32 0 0 0 32 0 151 Sphagnum fallax 26 25 0 0 0 25 0 Algae 152 Bathycoccus prasinos 0 0 0 0 0 0 0 153 Chlamydomonas reinhardtii 0 0 0 0 0 0 0 154 Chlorella sp. NC64A 0 0 0 0 0 0 0 155 Coccomyxa sp. 0 0 0 0 0 0 0 156 Dunaliella salina 0 0 0 0 0 0 0 157 Klebsormidium flaccidum 3 0 0 0 0 0 0 158 Micromonas pusilla 0 0 0 0 0 0 0 159 Ostreococcus lucimarinus 0 0 0 0 0 0 0 160 Volvox carteri 0 0 0 0 0 0 0 1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026 Table 2

1027 Interactome partners of NAC TFs in plants.

1028

NAC Experimental Co-expression Text mining Interactions TFs Interactions NAC1 RNS1, NAC024, NAC095, ARV1, AT2G01410, AT3G10260, AT1G60380, AT1G60340 AT1G17080 NAC2 ERD14 NAC32, NAC102, NAC32, NAC102 DREB2A NAC3 *** **** NTL NAC4 *** **** UBC30 NAC5 **** **** CYP96A2, MYB NAC7 VND7 XCP1, XCP2 VND7, MYB46 NAC8 *** ATM, ATR ATM, ATR NAC10 *** MYB83, MYB63 MYB83, MYB85, MYB46, MY63, MYB58, MYB52, MYB69, KNAT NAC11 **** **** NAC95 NAC12 * IRX1 MYB46, MYB83, MYB58, MYB63, IRX9, APL, KNAT7 NAC13 RCD1 AOX1A, RCD1 AOX1A, RCD1, NAC88 NAC14 ASG2 ASG2, ASG8, AT1G61900 NAC16 NYE, NACA5, NAC17 NAC88, AT5G13610 NAC18 GAI NAM, NAC NAC19 ZFHD1, TCP20, NAC32, ERD1 ZFHD1, TCP20, CPL1, TCP8, NAC32, RHA1A, CPL1, TCP8, RHA2A, ERD1 NAC32, RHA1A, RHA2A NAC20 AT3G43430, SHR, TMO6,DOF6, SHR, TMO6,DOF6, SHR, PLT2, AT1G64620, PHB, PLT2, PLT2 AT3G43430 MYB59, HB23, HB30 NAC23 **** ***** NAC95, AT3G01030, AT5G27880, AT5G01860, MYB64 NAC24 **** ***** NAC95, NAC47 NAC25 **** At1g75910, GRP20, At1g75910, GRP20, CYP86C4 CYP86C4 NAC26 VND7 VND7, MYB83, VND7, MYB46, MYB85, MYB83, XCP1 XCP1, AT4G08160 NAC028 ***** ******* TOM2B, DBP1, PDLP2, TOM2A NAC29 NAC6, GRL, NAC6, HAI1 NAC6, HAI1, SAG12, PI IAA14, NAC32 HAI1, NAC019, ATAF1, HAI1, NAC102, NAM, NAC19ATAF1 ABI1, NAM, NAC019, GSTU7, RVE2, PYL4 NAC36 ***** AT5G52760, AT5G42050

31 bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

XBAT34, AT5G52750, SOBIR1, RING1, WRKY53, WRKY46, SARD1, NAC38 BRM MYB69, CIPK4, AT4G29770, AIP2, SDE3 ABCA8 NAC41 NAC83 NAC83, NAC83, GSTF3, AT1G12810 AT1G12810 NAC42 **** CYP71A12, CYP71A12, GSTU10, AT5G38900, GSTU10, AT5G38900, CYP71B6 NAC44 **** **** AT1G54890, NAC90 NAC45 HB52, NAC97 NAC97 CYP71B34, WAK5, NAC97 NAC46 RCD1, BRM CYP89A9, RCD1, AT1G78040, bHLH11, AT4G11910 NAC47 *** HAI1, Rap2.6L, NAC5, NAC24, HAI1, AT1G60380 NAC6 NAC48 **** ***** CYP89A9, STAY-GREEN2 NAC49 **** ***** ERF115, WOX5, LBD19 NAC50 JMJ14, NAC052, NAC52, JMJ14 JMJ14, PPR, NAC52, AT5G41650, CYP71A25 GAI, TPL NAC52 JMJ14, NAC50 JMJ14, PPR, UBP14 JMJ14, NAC50, PPR, CRCK2, PPD6, MFDX1, CYP71A25 NAC53 **** AT5G25930, NTL, AT5G25930, AT3G25610, UGT73B5, NAC55 ZFHD1, HAI1, ERD1, AT2G31945, ZFHD1, ERD1, HAI1, ABF2, bZIP, MYC2 F2P16.14 MYB2 NAC57 ***** ***** MYB19, AT3G58090, AT1G07730, AT4G13580, AT3G13650 NAC58 ***** RWP1, ABCG6, PPR, RWP1, ABCG6, MYB86, MYB26 CYP86A1 NAC60 **** OLEO1 NACA5, AT3G52350, RINL, AT1G65240, NTL NAC61 **** NAC90, ACS4, NAC44, LEA, NAC85, NAC95, NAC90, NAC62 **** BZIP60, WRKY33, BZIP60, WRKY33, TIP TIP, SZF1, CPK32, CPK28, NHL3 NAC63 ***** ****** LRR, NAC95, ATPMEPCRD, NAC64 ***** ***** AT3G59880, AT5G50540, AT2G44010, sks16, SKS6 NAC66 ***** ***** MYB26, MYB46, MYB83, MYB85, MYB63, MYB58, KNAT7, WRKY12 NAC67 ***** **** NAM, AT1G78040, NAC95 NAC69 **** ***** NAC95, CYP96A2, NAM NAC71 **** WNK, TM6, Rap2.6L, AT2G41870, RAP2.4 AT1G64625 NAC73 **** MYB46, MYB83, MYB46, MYB83, IRX1, IRX3, MYB63, CESA4

IRX1, IRX3, CESA4 NAC74 F2P16.14, DSEL, scpl31, SCRL20, F-ox/LLR, sks11 TOPLESS, BRM HXXXD type NAC75 RING/U-box, ZF ****** ERF16, UTr7, CML, TPL/TPR, domain NAC76 VND7, NAC83 **** VND7, NAC83, UBQ, MYB46 NAC77 ****** ****** PIP1-5 NAC80 BRM ***** PPR, TT7, 4CL3, BRM NAC82 SRO1, RCD1 ***** UBX, WW NAC83 VND7, NAC41, ***** VND7, NAC41, CUC2, VND1, NAC105, CUC2, VND1, NAC76, MYB83, MYB46 NAC105, NAC76, NAC101, NAC1 NAC84 **** EDF3 ZFP10, Delta9, EDF3, SPT16, GS1 NAC85 **** **** LEA, PUP4, NAC90, NAC61, XERO1 NAC87 **** **** SWAP, WRKY36, TIR-NBS, NBS-LRR, BHLH11 NAC88 **** **** UBC18, NAC17, NAC13, NAC53 NAC89 PAS1, MYB, TI1, ***** PAS1, maMYB, BZIP60, BZIP28 TSPO, TIP2.2, TIP3.1 NAC90 ***** AT3G57460, DTA4, CHI, NAC44, NAC85, LEA MPK11 NAC94 ***** ***** MC5, D111, RML, BAG6, LCAT3, AATP1, BZIP28 NAC95 ***** NAC24, NAM NAC23, NAM, NAC24, MAY64, NAC69 NAC96 T21F11.18 ***** ABF2, Dna-J, TOPLESS, NAC97 NAC45, LRR, ***** ****** BRM NAC100 ***** ***** AT4G27850, AT1G26410, GRP20, TT7, 4CL3, NAC101 RPA2, VND7, VR- ***** NVD7, NAC83, XCP1, UBQ, RNS3 NAC, NAC83 NAC102 **** ATAF1, tolB, ATAF1, NAC32 NAC32, RHL41, ZAT6, UGT73B2 NAC103 **** ***** BZIP60, BZIP28, D111, CLPTM1, NAC44 NAC105 VND7, NAC83, ***** VND7, GH, NAC83, UBQ, LAC1, MYB46, RIC4 1029

1030

1031

1032

1033

1034 Table 3

1035 Codon usage of NAC TFs in plants.

1036

Codons Codon Codon absent Average Highest Name of the species with present in in No. of abundance no. of highest no. of codons No. of species of codons codons species AAA (K) 126 20 4.77 9.9 Glycine soja AAG (K) 146 0 10.75 24.2 Sphagnum fallax AAC (N) 144 2 3.66 14.2 Beta vulgaris AAU (N) 127 19 9.25 20.5 Spinacia oleracea ACA (T) 139 7 2.33 15.2 Citrus sinensis ACC (T) 137 9 2.4 17 Amborella trichopoda ACG (T) 20 126 5.91 13 Dorcoceras hygrometricum ACU (T) 146 0 7.42 16.6 Sesamum indicum AGA (R) 146 0 10.92 24.3 Klebsormidium flaccidum AGG (R) 146 0 4.12 18.8 Amborella trichopoda CGA (R) 19 127 5.22 13.9 Linum usitatissimum CGC (R) 19 127 2.47 6 Linum usitatissimum CGG (R) 19 127 3.93 8.6 Citrullus lanatus CGU (R) 19 127 2.06 4.7 Linum usitatissimum AGC (S) 143 3 3.54 24.2 Beta vulgaris AGU (S) 144 2 1.83 5.2 Dorcoceras hygrometricum UCC (S) 141 5 4.51 12.3 Aegilops tauschii UCG (S) 20 126 2.64 6.4 Dorcoceras hygrometricum UCU (S) 146 0 4.65 30.5 Humulus lupulus UCA (S) 139 7 5.09 15.1 Morus notabilis AUA (I) 124 22 4.80 15.3 Sphagnum fallax AUC (I) 146 0 5.10 16.7 Sphagnum fallax AUU (I) 126 20 8.71 15.9 Spinacia oleracea AUG (M) 146 0 7.81 22.8 Sphagnum fallax CAA (Q) 146 0 5.31 15.4 Fragaria vesca CAG (Q) 20 126 13.3 22.6 Linum usitatissimum CAC (H) 20 126 6.64 10.9 Beta vulgaris CAU (H) 144 2 4.45 9.7 Setaria viridis CCA (P) 20 126 11.09 16.3 Dorcoceras hygrometricum CCC (P) 20 126 14.18 19.2 Amborella trichopoda CCG (P) 20 126 5.10 11.1 Dorcoceras hygrometricum CCU (P) 146 0 8.00 24.7 Klebsormidium flaccidum CUA (L) 143 3 5.83 28.3 Sphagnum fallax CUC (L) 123 23 5.74 23.6 Sphagnum fallax CUG (L) 142 4 5.87 43.9 Sphagnum fallax CUU (L) 145 1 5.94 32.6 Sphagnum fallax UUG (L) 125 21 5.94 24.4 Sphagnum fallax UAA (L) 124 22 5.37 17.2 Sphagnum fallax GAA (E) 146 0 4.62 27 Klebsormidium flaccidum GAG (E) 145 1 5.54 18.1 Sphagnum fallax

GAC (D) 145 1 5.05 14.9 Beta vulgaris GAU (D) 144 2 5.86 21.7 Spinacia oleracea GCA (A) 135 11 5.49 18.5 Citrus sinensis GCC (A) 130 16 5.05 15 Amborella trichopoda GCG (A) 20 126 4.64 11.2 Dorcoceras hygrometricum GCU (A) 146 0 4.65 31.1 Setaria viridis GGA (G) 146 0 4.63 27.5 Setaria viridis GGC (G) 141 5 5.41 17.1 Amborella trichopoda GGG (G) 145 1 2.7 6.7 Elaeis guineensis GGU (G) 145 1 2.40 5.9 Elaeis guineensis GUA (V) 140 6 1.46 3.5 Sphagnum fallax GUC (V) 123 23 0.93 2 Morus notabilis GUG (V) 142 4 4.35 11.8 Beta vulgaris GUU (V) 143 3 5.38 16.6 Klebsormidium flaccidum UAC (Y) 138 8 3.84 10.1 Morus notabilis UAU (Y) 126 20 6.23 14.8 Solanum melongena UGG (W) 147 0 3.89 14.5 Vitis vinifera UGC (C) 143 3 5.14 15.6 Oropetium thomaeum UGU (C) 145 1 3.9 9.6 Zoysia matrella UUC (F) 146 0 4.60 25.4 Picea glauca UUU (F) 126 20 10.67 19.2 Sphagnum fallax 1037

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052 Table 4. Substitution rate of NAC TFs of plants.

A T C G A T C G A T C G A T C G Actinidia chinensis Aegilops tauschi Aethionema arabicum Amaranthus hypochondriacus A - 10.6 4.26 10.8 - 10.3 4.7 12.3 - 11.4 4.3 10.1 - 11.6 4.83 10.4 2 3 6 1 2 3 5 9 5 T 8.34 - 4.34 5.38 7.78 - 4.3 5.96 9.01 - 3.54 5.7 8.54 - 3.45 5.16 7 C 8.34 10.8 - 5.38 7.78 9.62 - 5.96 9.01 9.39 - 5.7 8.54 8.35 - 5.16 3 G 16.79 10.6 4.26 - 16.09 10.3 4.7 - 16.0 11.4 4.3 - 17.3 11.6 4.83 - 2 6 1 4 3 2 9 Amborella trichopoda Ananas comosus Aquilegia coerulea Arabidopsis halleri A - 3.99 10.9 14.1 - 3.73 10.9 15.3 - 11 4.33 11.1 - 10.9 4.15 11.0 8 4 5 6 1 3 7 T 8.17 - 5.41 10.7 7.66 - 5.91 10.3 8.67 - 3.44 5.32 8.98 - 3.3 5.73 2 C 8.17 1.97 - 10.7 7.66 2.01 - 10.3 8.67 8.73 - 5.32 8.98 8.71 - 5.73 2 G 10.8 3.99 10.9 - 11.3 3.73 10.9 - 18.0 11 4.33 - 17.3 1.93 4.15 - 8 9 5 9 4 Arabidopsis lyrata Arabidopsis thaliana Arabis alpina Arachis duranensis A - 4.03 10.6 14.3 - 4.06 10.4 14.8 - 11.0 4.25 10.6 - 11.1 4.43 10.3 5 6 4 2 7 T 8.63 - 4.51 10.6 8.41 - 4.65 10.5 8.88 - 3.63 5.67 8.76 - 3.82 5.37 4 2 C 8.63 1.71 - 10.6 8.41 1.82 - 10.5 8.88 9.42 - 5.67 8.76 9.62 - 5.37 4 2 G 11.6 4.03 10.6 - 11.8 4.06 10.4 - 16.6 11.0 4.25 - 16.7 11.1 4.43 - 3 9 3 4 9 7 Arachis hypogaea Arachis ipaensis Artemisia annua Azadirachta indica A - 4 11.0 14.2 - 3.93 10.8 14.6 - 10.8 4.08 10.9 - 10.9 4.22 10.3 7 1 5 3 5 3 8 4 T 8.16 - 5.53 10.2 8.39 - 4.95 10 8.24 - 4.22 5.62 8.91 - 3.82 5.6 4 C 8.16 2 - 10.2 8.39 1.79 - 10 8.24 11.2 - 5.62 8.91 9.95 - 5.6 4 4 G 11.3 4 11.0 - 12.2 3.93 10.8 - 16.0 10.8 4.08 - 16.4 10.9 4.22 - 2 7 8 5 1 5 4 8 Beta vulgaris Boechera stricta Brachypodium distachyon Brachypodium stacei A - 3.85 10.9 14.3 - 11.0 4.23 10.8 - 10.6 4.93 12.5 - 10.5 4.96 12.6 5 8 2 6 7 2 5 T 8.62 - 4.72 10 8.77 - 3.57 5.58 7.75 - 4.05 6.19 7.68 - 4.16 6.21 C 8.62 1.66 - 10 8.77 9.29 - 5.58 7.75 8.71 - 6.19 7.68 8.83 - 6.21 G 12.4 3.85 10.9 - 17.0 11.0 4.23 - 15.7 10.6 4.93 - 15.6 10.5 4.96 - 5 9 2 4 3 2 Brassica napus Brassica oleracea Brassica rapa Cajanus cajan A - 10.8 4.26 10.7 - 10.8 4.29 10.7 - 3.98 10.7 14.4 - 11.3 4.53 10.3

3 1 3 2 3 7 1 T 8.61 - 4.01 5.57 8.7 - 3.91 5.57 8.48 - 4.63 10.6 8.63 - 3.9 5.53 C 8.61 10.1 - 5.57 8.7 9.87 - 5.57 8.48 1.72 - 10.6 8.63 9.73 - 5.53 8 G 16.5 10.8 4.26 - 16.7 10.8 4.29 - 11.5 3.98 10.7 - 16.0 11.3 4.53 - 7 3 3 3 8 3 7 1 Camelina sativa Cannabis sativa Capsella grandiflora Capsella rubella A - 3.88 10.4 14.9 - 11.1 4.35 10.4 - 12.3 4.92 9.49 - 11.1 4.35 10.4 4 7 9 4 9 8 T 8.41 - 4.9 10.4 9.25 - 3.24 5.72 9.91 - 2.41 6.34 8.81 - 3.69 5.65 6 C 8.41 1.82 - 10.4 9.25 8.33 - 5.72 9.91 6.2 - 6.34 8.81 9.48 - 5.65 6 G 11.9 3.88 10.4 - 16.9 11.1 4.35 - 14.8 12.3 4.92 - 16.3 11.1 4.35 - 9 4 7 7 3 4 3 9 1053

Capsicum annuum Carica papaya Castanea mollissima Catharanthus roseus A - 10.9 3.95 10.8 - 10.4 4.05 11.4 - 11.3 4.53 10.0 - 10.5 4.13 11.3 5 3 9 1 3 4 5 T 8.91 - 3.47 5.49 8.39 - 3.98 5.47 8.98 - 3.75 5.73 8.6 - 3.82 5.69 C 8.91 9.62 - 5.49 8.39 10.2 - 5.47 8.98 9.37 - 5.73 8.6 9.76 - 5.69 4 G 17.5 10.9 3.95 - 17.6 10.4 4.05 - 15.7 11.3 4.53 - 17.1 10.5 4.13 - 1 5 3 3 2 1 4 4 Chenopodium quinoa Cicer arietinum Citrullus lanatus Citrus clementina A - 11.2 4.48 10.4 - 4.05 10.9 13.8 - 3.95 10.8 14.6 - 3.9 10.5 14.7 6 1 2 2 6 2 T 8.77 - 3.76 5.67 8.93 - 4.36 10.1 8.25 - 5.19 10.2 8.33 - 5.26 10.2 7 4 9 C 8.77 9.4 - 5.67 8.93 1.62 - 10.1 8.25 1.89 - 10.2 8.33 1.94 - 10.2 7 4 9 G 16.1 11.2 4.48 - 12.1 4.05 10.9 - 11.7 3.95 10.8 - 11.9 3.9 10.5 - 6 1 1 7 2 2 6 Citrus sinensis Coffea canephora Cucumis melo Cucumis sativus A - 3.93 10.4 14.3 - 11.2 4.64 10.3 - 11.3 4.53 10.2 - 4.18 10.9 14.1 9 7 2 1 1 7 2 T 8.39 - 5.72 10.1 8.63 - 4.03 5.76 8.5 - 4.07 5.39 8.45 - 4.75 10.2 2 C 8.39 2.14 - 10.1 8.63 9.75 - 5.76 8.5 10.1 - 5.39 8.45 1.81 - 10.2 2 7 G 11.9 3.93 10.4 - 15.4 11.2 4.64 - 16.0 11.3 4.53 - 11.6 4.18 10.9 - 1 9 2 2 9 1 9 7 Daucus carota Dianthus caryophyllus Dichanthelium Dorcoceras oligosanthes hygrometricum A - 4.04 10.9 13.8 - 3.93 10.7 14.2 - 10.5 4.97 12.6 - 4.04 10.8 14.1 9 4 6 5 6 5 4 T 8.52 - 5.16 10.3 8.57 - 5.02 10.2 7.49 - 4.32 5.97 8.47 - 5.04 10.3 4 5

37 bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

C 8.52 1.89 - 10.3 8.57 1.84 - 10.2 7.49 9.16 - 5.97 8.47 1.88 - 10.3 4 5 G 11.3 4.04 10.9 - 11.9 3.93 10.7 - 15.8 10.5 4.97 - 11.6 4.04 10.8 - 7 9 1 4 8 5 2 5 Elaeis guineensis Eragrostis tef Eucalyptus camaldulensis Eucalyptus grandis A - 3.85 10.7 15.2 - 10.7 4.7 11.4 - 10.8 4.28 10.8 - 3.95 10.6 14.9 9 8 2 8 4 1 8 4 T 7.88 - 5.23 10.6 8.34 - 3.99 6.09 8.47 - 4.13 5.7 7.87 - 5.87 10.3 3 1 C 7.88 1.86 - 10.6 8.34 9.12 - 6.09 8.47 10.4 - 5.7 7.87 2.17 - 10.3 3 4 1 G 11.3 3.85 10.7 - 15.7 10.7 4.7 - 16.0 10.8 4.28 - 11.4 3.95 10.6 - 3 9 2 2 5 4 8 Eutrema salsugineum Fragaria vesca Fragaria x ananassa Genlisea aurea A - 11.0 4.27 10.6 - 10.8 4.49 10.6 - 10.2 4.07 11.1 - 10.5 3.98 11.5 4 7 5 7 2 9 8 T 8.8 - 3.67 5.62 8.64 - 3.96 5.4 8.26 - 4.48 5.34 9.08 - 3.34 6.52 C 8.8 9.49 - 5.62 8.64 9.56 - 5.4 8.26 11.2 - 5.34 9.08 8.79 - 6.52 4 G 16.7 11.0 4.27 - 17.0 10.8 4.49 - 17.3 10.2 4.07 - 16.1 10.5 3.98 - 4 6 5 2 2 2 Glycine max Glycine soja Gossypium arboreum Gossypium hirsutum A - 11.4 4.62 10.1 - 11.5 4.72 10.0 - 11.0 4.39 10.5 - 11.0 4.4 10.5 3 8 3 7 5 6 6 1 T 8.78 - 3.73 5.59 8.98 - 3.55 5.68 8.64 - 3.89 5.5 8.6 - 3.98 5.55 C 8.78 9.24 - 5.59 8.98 8.67 - 5.68 8.64 9.8 - 5.5 8.6 10.0 - 5.55 2 G 16.0 11.4 4.62 - 15.9 11.5 4.72 - 16.5 11.0 4.39 - 16.2 11.0 4.4 - 1 3 2 3 8 5 7 6 Gossypium raimondii Helianthus annuus Hordeum vulgare Humulus lupulus A - 11.2 4.52 10.2 - 10.3 4.31 10.7 - 10.7 4.8 11.6 - 11.2 4.63 10.5 8 2 9 9 4 1 4 1 2 T 8.85 - 3.77 5.62 8.23 - 4.74 5.46 8.41 - 3.8 6.48 8.7 - 3.68 5.37 2 C 8.85 9.39 - 5.62 8.23 11.4 - 5.46 8.41 8.53 - 6.48 8.7 8.92 - 5.37 2 G 16.0 11.2 4.52 - 16.2 10.3 4.31 - 15.1 10.7 4.8 - 17.0 11.2 4.63 - 9 8 8 9 2 4 1 6 1 Ipomoea trifida Jatropha curcas Juglans regia Kalanchoe laxiflora A - 10.7 4.47 10.9 - 11.1 4.34 10.6 - 11.0 4.4 10.8 - 11.5 4.93 9.82 9 4 9 6 9 5 2 2 T 8.33 - 4.22 5.6 8.68 - 3.66 5.5 8.45 - 3.9 5.64 8.72 - 4.03 5.74 3 C 8.33 10.1 - 5.6 8.68 9.44 - 5.5 8.45 9.79 - 5.64 8.72 9.4 - 5.74 9 G 16.2 10.7 4.47 - 16.8 11.1 4.34 - 16.2 11.0 4.4 - 14.9 11.5 4.93 - 7 9 3 9 2 9 5 2 2 Kalanchoe marnieriana Klebsormidium flaccidum Lactuca sativa Leersia perrieri A - 12.0 5.24 9.86 - 7.73 4.03 13.0 - 11.3 4.5 9.9 - 10.3 4.57 13.2

8 8 9 7 1 7 T 9.37 - 2.86 6.19 6.67 - 7.19 5.04 9.13 - 3.6 5.91 7.33 - 4.28 5.82 7 C 9.37 6.59 - 6.19 6.67 13.7 - 5.04 9.13 9.16 - 5.91 7.33 9.66 - 5.82 8 G 14.9 12.0 5.24 - 18.2 7.73 4.03 - 15.2 11.3 4.5 - 16.7 10.3 4.57 - 3 8 8 8 9 7 3 1 Linum usitatissimum Lotus japonicus Malus domestica Manihot esculenta A - 3.72 10.3 15.8 - 10.3 3.72 12.6 - 10.6 4.2 11.2 - 11.2 4.54 10.4 9 2 1 4 5 3 1 2 8 T 7.89 - 5.6 10.0 8.2 - 3.43 5.44 8.18 - 4.1 5.24 8.65 - 3.82 5.57 9 7 C 7.89 2.01 - 10.0 8.2 9.51 - 5.44 8.18 10.5 - 5.24 8.65 9.45 - 5.57 9 G 12.3 3.72 10.3 - 19.0 10.3 3.72 - 17.5 10.6 4.2 - 16.2 11.2 4.54 - 7 9 7 1 5 3 9 2 Medicago truncatula Mimulus guttatus Morus notabilis Musa acuminata A - 11.2 4.3 10.1 - 10.7 4.02 10.5 - 11.1 4.5 10.6 - 10.9 4.62 10.8 4 9 2 1 6 4 4 3 9 T 8.9 - 3.68 5.4 8.91 - 3.91 5.67 8.54 - 3.8 5.32 8.14 - 4.42 5.9 C 8.9 9.62 - 5.4 8.91 10.4 - 5.67 8.54 9.34 - 5.32 8.14 10.4 - 5.9 2 7 G 16.7 11.2 4.3 - 16.5 10.7 4.02 - 17.1 11.1 4.5 - 15.0 10.9 4.62 - 9 4 1 2 6 4 5 3 Nelumbo nucifera Nicotiana benthamiana Nicotiana sylvestris Nicotiana tabacum A - 10.8 4.4 10.9 - 10.8 4.11 10.3 - 10.8 4.2 10.4 - 11.2 4.31 10.2 8 8 9 8 6 1 T 8.43 - 4.04 5.56 8.82 - 3.89 5.38 8.87 - 3.8 5.44 9.07 - 3.57 5.55 6 C 8.43 9.99 - 5.56 8.82 10.2 - 5.38 8.87 9.87 - 5.44 9.07 9.28 - 5.55 9 G 16.5 10.8 4.4 - 17.0 10.8 4.11 - 16.9 10.8 4.2 - 16.6 11.2 4.31 - 5 8 3 8 8 8 6 9 Nicotiana tomentosiformis Ocimum tenuiflorum Oropetium thomaeum Oryza brachyantha A - 11.0 4.3 10.4 - 11.0 4.32 10.5 - 10.5 4.8 12.4 - 10.5 4.85 12.9 5 3 2 9 2 2 1 1 8 T 8.93 - 3.64 5.43 8.44 - 4.15 5.68 7.46 - 4.5 5.93 7.3 - 4.37 5.92 6 C 8.93 9.36 - 5.43 8.44 10.5 - 5.68 7.46 9.97 - 5.93 7.3 9.47 - 5.92 8 G 17.1 11.0 4.3 - 15.7 11.0 4.32 - 15.6 10.5 4.8 - 16.0 10.5 4.85 - 6 5 4 2 1 2 2 1 1 1055

Oryza glaberrima Oryza glumaepatula Oryza longistaminata Oryza meridionalis A - 10.65 5.01 12.8 - 10.7 5 12.4 - 10.6 4.7 12.1 - 10.4 4.87 13.3 6 1 7 9 4 1 2 T 7.52 - 4.08 6.14 7.61 - 4.18 6.16 7.96 - 4.0 6.09 7.43 - 4.1 6.12 3

39 bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

C 7.52 8.69 - 6.14 7.61 8.96 - 6.16 7.96 9.09 - 6.09 7.43 8.79 - 6.12 G 15.7 10.65 5.01 - 15.4 10.7 5 - 15.8 10.6 4.7 - 16.1 10.4 4.87 - 3 2 1 2 9 4 4 2 Oryza nivara Oryza punctata Oryza rufipogon Oryza sativa A - 10.64 5.01 12.4 - 10.7 4.94 12.3 - 10.6 5.0 12.8 - 10.8 5.12 12.2 9 1 7 7 4 4 6 2 T 7.6 - 4.25 6.17 7.66 - 4.2 6.23 7.47 - 4.1 6.18 7.64 - 4.21 6.33 4 C 7.6 9.01 - 6.17 7.66 9.12 - 6.23 7.47 8.76 - 6.18 7.64 8.94 - 6.33 G 15.3 10.6 5.01 - 15.2 10.7 4.94 - 15.5 10.6 5.0 - 17.7 10.8 5.12 - 9 2 1 3 7 4 3 6 Panicum hallii Panicum virgatum Petunia axillaris Petunia inflata A - 10.66 5 12.6 - 10.4 5.02 12.7 - 10.0 4.1 10.0 - 11.1 4.28 10.2 3 2 3 8 7 3 8 T 7.75 - 3.97 6.32 7.53 - 4.39 6.14 8.77 - 4.0 5.35 8.83 - 3.79 5.41 5 C 7.75 8.47 - 6.32 7.53 9.12 - 6.14 8.77 10.6 - 5.35 8.83 9.86 - 5.41 8 G 15.4 10.66 5 - 15.6 10.4 5.02 - 16.5 10.0 4.1 - 16.7 11.1 4.28 - 8 2 3 8 7 3 Phaselous vulgaris Phoenix dactylifera Phyllostachys heterocycla Physcomitrella patens A - 11.4 4.62 10.2 - 10.7 4.44 11.1 - 11.0 4.9 11.6 - 10.6 4.64 10.7 4 1 6 3 7 1 7 T 8.75 - 3.78 5.69 8.2 - 4.31 5.71 8.1 - 3.9 6.27 8.1 - 4.77 5.53 4 C 8.75 9.32 - 5.69 8.2 10.3 - 5.71 8.1 8.73 - 6.27 8.1 10.9 - 5.53 9 2 G 15.7 11.4 4.62 - 16.0 10.7 4.44 - 14.9 11.0 4.9 - 15.7 10.6 4.64 - 5 1 9 3 7 7 1 Picea abies Picea glauca Picea sitchensis Pinus taeda A - 10.82 4.54 10.2 - 10.8 4.61 10.3 - 10.3 4.4 10.5 - 10.0 4.37 11.6 9 5 5 5 5 8 8 7 T 8.53 - 4.42 5.48 8.42 - 4.47 5.42 8.02 - 5.1 5.12 8.18 - 4.39 5.35 1 C 8.53 10.53 - 5.48 8.42 10.5 - 5.42 8.02 11.8 - 5.12 8.18 10.1 - 5.35 2 7 1 G 16 10.82 4.54 - 16.0 10.8 4.61 - 16.5 10.3 4.4 - 17.8 10.0 4.37 - 8 5 6 5 5 6 8 Populus euphratica Populus trichocarpa Prunus mume Prunus persica A - 10.86 4.53 10.8 - 10.9 4.52 10.6 - 11.1 4.4 10.6 - 11.3 4.53 10.2 5 8 8 2 3 5 8 3 T 8.33 - 4.15 5.31 8.47 - 4.01 5.35 8.91 - 3.4 5.55 8.95 - 3.55 5.6 9 C 8.33 9.95 - 5.31 8.47 9.74 - 5.35 8.91 8.76 - 5.55 8.95 8.92 - 5.6 G 17 10.86 4.53 - 16.9 10.9 4.52 - 17.0 11.1 4.4 - 16.3 11.3 4.53 - 3 8 7 2 3 6 8 Pseudotsuga menziesii Pyrus bretschneideri Raphanus raphanistrum Raphanus sativus A - 10.74 4.44 9.98 - 11.1 4.51 10.7 - 11.0 4.4 10.5 - 10.9 4.35 10.7 8 7 1 6 1

T 8.49 - 4.79 5.68 8.57 - 3.77 5.47 8.72 - 3.8 5.6 8.47 - 4.06 5.52 5 C 8.49 11.59 - 5.68 8.57 9.26 - 5.47 8.72 9.7 - 5.6 8.47 10.2 - 5.52 3 G 14.9 10.74 4.44 - 16.8 11.1 4.51 - 16.3 11.0 4.4 - 16.4 10.9 4.35 - 3 9 6 7 4 6 1056

Ricinus communis Saccharum officinarum Salix purpurea Salvia miltiorrhiza A - 10.87 4.28 11.0 - 9.95 4.48 12.7 - 10.8 4.5 10.9 - 11.2 4.64 10.6 5 8 5 8 7 3 T 8.56 - 3.76 5.48 8.25 - 3.94 6.61 8.24 - 4.1 5.32 8.49 - 3.89 5.7 6 C 8.56 3.76 - 5.48 8.25 8.76 - 6.61 8.24 10.0 - 5.32 8.49 9.44 - 5.7 4 G 17.2 10.87 4.28 - 15.9 9.95 4.48 - 17.0 10.8 4.5 - 15.8 11.2 4.64 - 7 5 1 5 2 7 Selaginella moellendorffii Sesamum indicum Setaria italica Setaria viridis A - 10.76 4.13 11.6 - 11.3 4.82 10.1 - 10.8 5.1 12.2 - 10.6 5.05 12.4 5 7 2 3 9 5 T 8.65 - 3.52 6.3 8.63 - 4.03 5.65 7.69 - 4.1 6.22 7.67 - 4.22 6.1 2 C 8.65 9.15 - 6.3 8.63 9.45 - 5.65 7.69 8.67 - 6.22 7.67 8.87 - 6.1 G 16.0 10.76 4.13 - 15.5 11.3 4.82 - 15.1 10.8 5.1 - 15.6 10.6 5.05 - 1 4 9 2 3 5 Sisymbrium irio Solanum lycopersicum Solanum melongena Solanum pennellii A - 10.74 4.29 11.0 - 11.1 4.12 10.1 - 10.7 4.0 10.6 - 11.0 4.11 10.2 5 7 3 2 4 7 7 5 T 8.45 - 3.96 5.37 9.1 - 3.58 5.45 8.67 - 3.9 5.3 8.96 - 3.7 5.39 5 C 8.45 9.91 - 5.37 9.1 9.7 - 5.45 8.67 10.4 - 5.3 8.96 9.95 - 5.39 8 G 17.3 10.74 4.29 - 16.9 11.1 4.12 - 17.4 10.7 4.0 - 17.0 11.0 4.11 - 7 7 6 2 4 3 7 Solanum pimpinellifolium Solanum tuberosum Sorghum bicolor Sphagnum fallax A - 11.4 4.23 9.8 - 10.9 4.13 10.3 - 10.6 5.0 12.2 - 10.9 4.92 10.6 6 4 4 7 4 5 5 T 9.3 - 3.48 5.58 8.83 - 3.86 5.38 7.76 - 4.2 6.21 8.03 - 4.69 5.7 3 C 9.3 9.38 - 5.58 8.83 10.2 - 5.38 7.76 8.88 - 6.21 8.03 10.4 - 5.7 4 4 G 16.3 11.4 4.23 - 16.9 10.9 4.13 - 15.3 10.6 5.0 - 15 10.9 4.92 - 3 8 6 1 4 7 5 Spinacia oleracea Spirodela polyrhiza Tarenaya hassleriana Thellungiella parvula A - 11.37 4.57 10.2 - 11.0 4.84 10.6 - 11.0 4.5 10.6 - 11.3 4.46 10.2 1 9 5 2 6 4 1 T 8.74 - 3.76 5.4 8.45 - 4.21 6.38 8.73 - 3.8 5.81 8.69 - 3.9 5.62 1 C 8.74 9.35 - 5.4 8.45 9.59 - 6.38 8.73 9.31 - 5.81 8.69 9.9 - 5.62

G 16.5 11.37 4.57 - 14.1 11.0 4.84 - 16.0 11.0 4.5 - 15.7 11.3 4.46 - 1 6 1 2 5 2 8 4 Theobroma cacao Trifolium pratense Triticum aestivum Triticum urartu A - 11.04 4.45 10.5 - 11.2 4.26 9.73 - 10.6 4.8 11.2 - 10.7 4.94 11.4 2 1 5 5 8 T 8.53 - 4.04 5.43 9.05 - 3.89 5.43 8.28 - 4.3 6.16 8.12 - 4.27 6.19 4 C 8.53 10.02 - 5.43 9.05 10.2 - 5.43 8.28 9.54 - 6.16 8.12 9.32 - 6.19 4 G 16.5 11.04 4.45 - 16.2 11.2 4.26 - 15.0 10.6 4.8 - 14.9 10.7 4.94 - 3 4 1 4 5 5 5 8 Utricularia gibba Vigna angularis Vigna radiata Vigna unguiculata A - 10.97 4.56 10.6 - 11.1 4.45 10.4 - 10.7 4.3 10.6 - 10.2 3.95 11.1 7 8 6 3 4 1 1 T 8.41 - 4.26 6 8.87 - 3.72 5.74 8.58 - 4.1 5.59 8.54 - 4.35 5.86 6 C 8.41 10.23 - 6 8.87 9.34 - 5.74 8.58 10.3 - 5.59 8.54 11.2 - 5.86 4 4 G 14.9 10.97 4.56 - 16.0 11.1 4.45 - 16.3 10.7 4.3 - 16.1 10.2 3.95 - 6 8 8 3 6 3 9 1 1057

Vitis vinifera Zea mays Ziziphus jujuba Zoysia japonica A - 11.27 4.64 10.2 - 10.7 5.14 12.1 - 11.0 5.5 10.7 - 10.8 5.02 12.0 1 7 3 4 1 3 8 T 8.69 - 3.98 5.74 7.82 - 4.15 6.34 8.6 - 3.8 5.47 7.87 - 4.04 6.18 4 C 8.69 9.68 - 5.74 7.82 8.69 - 6.34 8.6 9.33 - 5.47 7.87 8.72 - 6.18 G 15.4 11.27 4.64 - 14.9 10.7 5.14 - 16.8 11.0 5.5 - 15.3 10.8 5.02 - 5 1 7 4 3 4 8 3 Zoysia matrella Zoysia pacifica A - 10.65 5 12.2 - 10.4 4.88 12.6 9 1 7 T 7.73 - 4.21 6.11 7.57 - 4.35 6.04 C 7.73 8.98 - 6.11 7.57 9.28 - 6.04 G 15.5 10.65 5 - 18.8 10.4 4.88 - 4 9 1 1058

1059

1060

1061

1062

1063 Supplementary Data

1064 Supplementary Table 1

1065 Supplementary table showing different chimeric domains of NAC TFs.

1066 Supplementary Table 2

1067 NAC TFs showing the presence of novel functional domain along with NAC domains.

1068 Supplementary Table 3

1069 NAC TFs showing their involvement in different pathways and biological process.

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

1080

1081

1082

1083

1084

1085

1086

1087

1088

43 bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/608885; this version posted April 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.